Mass labels

ABSTRACT

Provided herein are sets of mass labels. Each mass label in a set includes 1) a mass marker moiety; 2) a mass normalisation moiety; and 3) a cleavable linker connecting the mass marker moiety to the mass normalisation moiety. Each mass marker moiety is characterised as having a mass different from that of all other mass marker moieties in the set as determined by mass spectrometry. Further, each mass normalisation moiety ensures that each mass label in the set has substantially the same mass as determined by mass spectrometry.

This application is the National Phase of International ApplicationPCT/GB01/01122 filed Mar, 14, 2001 which designated the U.S. and thatInternational Application was published under PCT Article 21(2) inEnglish.

This invention relates to useful compounds for labelling analytes,particularly biomolecules such as nucleic acids and proteins.Specifically this invention relates to methods of analysis by massspectrometry, using specific mass labels.

Various methods of labelling molecules of interest are known in the art,including radioactive atoms, fluorescent dyes, luminescent reagents,electron capture reagents and light absorbing dyes. Each of theselabelling systems has features which make it suitable for certainapplications and not others. For reasons of safety, interest innon-radioactive labelling systems has lead to the widespread commercialdevelopment of fluorescent labelling schemes particularly for geneticanalysis. Fluorescent labelling schemes permit the labelling of arelatively small number of molecules simultaneously, typically fourlabels can be used simultaneously and possibly up to eight. However thecosts of the detection apparatus and the difficulties of analysing theresultant signals limit the number of labels that can be usedsimultaneously in a fluorescence detection scheme.

More recently there has been development in the area of massspectrometry as a method of detecting labels that are cleavably attachedto their associated molecule of interest. In many molecular biologyapplications one needs to be able to separate the molecules of interestprior to analysis. Generally, liquid phase separations are performed.Mass spectrometry in recent years has developed a number of interfacesfor liquid phase separations, which make mass spectrometry particularlyeffective as a detection system for these kinds of applications. Untilrecently Liquid Chromatography Mass Spectrometry was used to detectanalyte ions or their fragment ions directly. However, for manyapplications such as nucleic acid analysis, the structure of the analytecan be determined from indirect labelling. This is advantageousparticularly with respect to the use of mass spectrometry becausecomplex biomolecules such as DNA have complex mass spectra and aredetected with relatively poor sensitivity. Indirect detection means thatan associated label molecule can be used to identify the originalanalyte, the label being designed for sensitive detection and having asimple mass spectrum. Simple mass spectra allow multiple labels to beused to analyse a plurality of analytes simultaneously.

PCT/GB98/00127 describes arrays of nucleic acid probes covalentlyattached to cleavable labels that are detectable by mass spectrometry,which identify the sequence of the covalently linked nucleic acid probe.The labelled probes of this application have the structure Nu-L-M whereNu is a nucleic acid covalently linked to L, a cleavable linker,covalently linked to M, a mass label. Preferred cleavable linkers inthis application cleave within the ion source of the mass spectrometer.Preferred mass labels are substituted poly-aryl ethers. This applicationdiscloses a variety of ionisation methods, and analysis by quadrupolemass analysers, time of flight (TOF) analysers and magnetic sectorinstruments as specific methods of analysing mass labels by massspectrometry.

PCT/GB94/01675 discloses ligands, and specifically nucleic acids,cleavably linked to mass tag molecules. Preferred cleavable linkers arephoto-cleavable. This application discloses Matrix Assisted LaserDesorption Ionisation (MALDI) TOF mass spectrometry as a specific methodof analysing mass labels by mass spectrometry.

PCT/US97/22639 discloses releasable non-volatile mass-label molecules.In preferred embodiments these labels comprise polymers, typicallybiopolymers which are cleavably attached to a reactive group or ligand,i.e. a probe. Preferred cleavable linkers appear to be chemically orenzymatically cleavable. This application discloses MALDI TOF massspectrometry as a specific method of analysing mass labels by massspectrometry.

PCT/US97/01070, PCT/US97/01046, and PCT/US97/01304 disclose ligands, andspecifically nucleic acids, cleavably linked to mass tag molecules.Preferred cleavable linkers appear to be chemically or photo-cleavable.These applications disclose a variety of ionisation methods and analysisby quadrupole mass analysers, TOF analysers and magnetic sectorinstruments as specific methods of analysing mass labels by massspectrometry.

The mass spectra generated for an analyte material are very sensitive tocontaminants. Essentially, any material introduced into the massspectrometer that can ionise will appear in the mass spectrum. Thismeans that for many analyses it is necessary to carefully purify theanalyte before introducing it into the mass spectrometer. For thepurposes of high throughput systems for indirect analysis of analytesthrough mass labels it would be desirable to avoid any unnecessarysample preparation steps. That is to say it would be desirable to beable to detect labels in a background of contaminating material and becertain that the peak that is detected does in fact correspond to alabel. The prior art does not disclose methods or compositions that canimprove the signal to noise ratio achievable in mass spectrometry baseddetection systems or that can provide confirmation that a mass peak in aspectrum was caused by the presence of a mass label.

For the purposes of detection of analytes after liquid chromatography orelectrophoretic separations, it is desirable that the labels usedminimally interfere with the separation process. If an array of suchlabels are used, it is desirable that the effect of each member of thearray on its associated analyte is the same as every other labels. Thisconflicts to some extent with the intention of mass marking which is togenerate arrays of labels that are resolvable in the mass spectrometeron the basis of their mass. Mass labels should preferably be resolved by4 daltons to prevent interference of isotope peaks from one label withthose of another label. This means that to generate 250 distinct masslabels would require labels spread over a range of about 1000 daltonsand probably more, since it is not trivial to generate large arrays oflabels separated by exactly 4 daltons. This range of mass will almostcertainly result in mass labels that will have a distinct effect on anyseparation process that precedes detection by mass spectrometry. It alsohas implications for instrument design, in that as the mass range overwhich a mass spectrometer can detect ions increases, the cost of theinstrument increases.

It is thus an object of this invention to solve the problems associatedwith the above prior art, and provide mass labels which can be detectedin a background of contamination and whose identity as mass labels canbe confirmed. Furthermore it is an object of this invention to providearrays of labels which can be resolved in a compressed mass range sothat the labels do not interfere as much with separation processes andwhich can be detected easily in a mass spectrometer that detects ionsover a limited range of mass to charge ratios.

It is also an object of this invention to provide methods of analysingbiomolecules which exploit the labels of this invention to maximisethroughput, signal to noise ratios and sensitivity of such assays,particularly in genetic analysis and more particularly 2-dimensional gelelectrophoresis which is used to analyse proteins.

Furthermore the design of the mass labels disclosed below allows asimplified tandem mass spectrometer to be designed for the purposes ofdetecting mass labels. The first mass analyser need only select alimited number of ions whose mass is relatively low. The second massanalyser need only detect a small number of fragmentation products.

Accordingly, the present invention provides a set of two or more masslabels, each label in the set comprising a mass marker moiety attachedvia a cleavable linker to a mass normalisation moiety, the mass markermoiety being fragmentation resistant, wherein the aggregate mass of eachlabel in the set may be the same or different and the mass of the massmarker moiety of each label in the set may be the same or different, andwherein in any group of labels within the set having a mass markermoiety of a common mass each label has an aggregate mass different fromall other labels in that group, and wherein in any group of labelswithin the set having a common aggregate mass each label has a massmarker moiety having a mass different from that of all other mass markermoieties in that group, such that all of the mass labels in the set aredistinguishable from each other by mass spectrometry.

The term mass marker moiety used in the present context is intended torefer to a moiety that is to be detected by mass spectrometry, whilstthe term mass normalisation moiety used in the present context isintended to refer to a moiety that is not necessarily to be detected bymass spectrometry, but is present to ensure that a mass label has adesired aggregate mass. The number of labels in the set is notespecially limited, provided that the set comprises a plurality oflabels. However, it is preferred if the set comprises two or more, threeor more, four or more, or five or more labels.

The present invention also provides an array of mass labels, comprisingtwo or more sets of mass labels as defined above, wherein the aggregatemass of each of the mass labels in any one set is different from theaggregate mass of each of the mass labels in every other set in thearray.

Further provided by the invention is a method of analysis, which methodcomprises detecting an analyte by identifying by mass spectrometry amass label or a combination of mass labels unique to the analyte,wherein the mass label is a mass label from a set or an array of masslabels as defined above.

The invention will now be described in further detail by way of exampleonly, with reference to the accompanying drawings, in which:

FIG. 1 shows a schematic layout of a triple quadrupole massspectrometer;

FIG. 2 shows ten fragments comprising five mass normalisation moieties(M₀-M₄), and five mass marker moieties (X₀-X₄), for forming a set oflabels according to the present invention, in which fluorine atomsubstituents are employed as mass adjuster moieties;

FIG. 3 shows a set of five labels according to the present invention,formed from the mass normalisation moieties and mass marker moieties ofFIG. 2;

FIG. 4 shows a set of five mass labels according to the presentinvention in which all the labels have a different mass, but in whichall the mass markers of the set have the same mass;

FIG. 5 shows an example of labelling an analyte such as anoligonucleotide with a combination of mass labels; such that the masslabel combination has a unique mass spectrum which identifies theanalyte;

FIG. 6 shows an array of sets of mass labels, each set having the samemass series modifying group (S) and being distinct from all other setsby virtue of the number of fluorine substituents on the base phenylgroup;

FIG. 7 shows an array of sets of mass labels, each set having the samemass series modifying group (S) and being distinct from all other setsby virtue of the number of phenyl ether units in the mass seriesmodifying group;

FIG. 8 illustrates the “mixing mode” embodiment of the presentinvention, showing 4 of the 8 possible unique mass spectra for allcombinations of three mass labels P, Q and R when present in relativequantities of 0 or 1;

FIG. 9 illustrates the “mixing mode” embodiment of the presentinvention, showing 8 of the 243 possible unique mass spectra for allcombinations of three mass labels P, Q, R, S and T when present inrelative quantities of 0, 1 or 2 (there are 81 possible spectra if Tremains constant as an internal standard);

FIG. 10 shows how larger sets of labels can be formed by enlarging themass normalisation and mass marker moieties to allow more scope forsubstitution—This set of labels has nine members, and uses fluorine atomsubstituents as mass adjuster moieties—a set of labels having at least 8members such as this is convenient for labelling all 256 4-mers in anarray of oligonucleotides using the mixing mode of the presentinvention;

FIG. 11 shows mass spectrum 1, which is a complete spectrum comprisingpeaks from all ions A⁺, B⁺, C⁺, and D₊;

FIG. 12 shows mass spectrum 2, which is a spectrum of A⁺ only, producedby selecting for A⁺ ions in a first quadrupole of the spectrometer (Q1);

FIG. 13 shows mass spectrum 3, which is a spectrum of a first ion A₁ ⁺(of the same mass/charge ratio as A⁺ and fragmentation products of A₁ ⁺,P⁺ and Q⁺;

FIG. 14 shows mass spectrum 4, which is a spectrum of a second ion A₂⁺(of the same mass/charge ratio as A⁺) and fragmentation products of A₂⁺, X⁺ and Y⁺;

FIG. 15 shows mass spectrum 5, which is a spectrum formed by selectingfor A⁺ ions when two types of such ions are present, A₁ ⁺ and A₂ ⁺;

FIG. 16 shows mass spectrum 6, which is a spectrum formed in a triplequadrupole spectrometer by selecting in Q1 for A⁺ ions when two types ofsuch ions are present (A₁ ⁺ and A₂ ⁺) inducing dissociation of theselected ions by collision in Q2, and selecting for a known collisionproduct of A₁ ⁺ (P⁺ in Q3—such a procedure allows resolution of A₁ ⁺ andA₂ ⁺;

FIG. 17 shows mass spectrum 7, which is a 2-dimensional spectrum of aset of five mass labels according to the present invention, in which amass MX is selected in Q1 (first dimension) and five distinct masses X₀,X₁, X₂, X₃, and X₄ are selected in Q3 (second dimension);

FIG. 18 shows mass spectrum 8, which is a 2-dimensional spectrum of aset of four mass labels according to the present invention, in whichfour distinct masses, M₀X₀, M₁X₀, M₂X₀, and M₃X₀, are selected in Q1(first dimension) and a single mass M₀ is selected in Q3 (seconddimension);

FIG. 19 shows mass spectrum 9, which is a 2-dimensional spectrum of aset of mass labels comprising labels formed from all combinations ofM₀-M₃ with X₀-X₃, in which seven distinct masses are selected in Q1(first dimension) and four distinct masses X₀-X₃ are selected in Q3(second dimension);

FIG. 20 shows a schematic of a typical cleavage process using the masslabels of the present invention and cleaving them from their analytesthermally, or using electrospray ionisation;

FIG. 21 shows a schematic of the selection procedures in 2-dimensionalmass spectrometry using a set of five mass labels according to thepresent invention.

FIG. 22 shows deuterated mass labels according to the present invention;

FIG. 23 shows further deuterated mass labels according to the presentinvention; and

FIG. 24 shows a theoretical spectrum for two samples of a peptide withthe sequence H₂N-gly-leu-ala-ser-glu-COOH (SEQ ID NO: 1), where eachsample is attached to one of the labels with the formulae shown in FIG.23.

In one preferred embodiment, the present invention provides a set ofmass labels as defined above, in which each label in the set has a massmarker moiety having a common mass and each label in the set has aunique aggregate mass. An example a set of labels of this first type isgiven in FIG. 4.

In an alternative, more preferred embodiment, each label in the set hasa common aggregate mass and each label in the set has a mass markermoiety of a unique mass. An example of a set of labels of this secondtype is given in FIG. 3.

The set of labels need not be limited to the two preferred embodimentsdescribed above, and may for example comprise labels of both types,provided that all labels are distinguishable by mass spectrometry, asoutlined above.

It is preferred that, in a set of labels of the second type, each massmarker moiety in the set has a common basic structure and each massnormalisation moiety in the set has a common basic structure, and eachmass label in the set comprises one or more mass adjuster moieties, themass adjuster moieties being attached to or situated within the basicstructure of the mass marker moiety and/or the basic structure of themass normalisation moiety. In this embodiment, every mass marker moietyin the set comprises a different number of mass adjuster moieties andevery mass label in the set has the same number of mass adjustermoieties.

Throughout this description, by common basic structure, it is meant thattwo or more moieties share a structure which has substantially the samestructural skeleton, backbone or core. This skeleton or backbone may befor example a phenyl ether moiety. The skeleton or backbone may comprisesubstituents pendent from it, or atomic or isotopic replacements withinit, without changing the common basic structure.

Typically, a set of mass labels of the second type referred to abovecomprises mass labels with the formula:M(A)_(y)-L-X(A)_(z)wherein M is the mass normalisation moiety, X is the mass marker moiety,A is a mass adjuster moiety, L is a cleavable linker, y and z areintegers of 0 or greater, and y+z is an integer of 1 or greater.Preferably M is a fragmentation resistant group, L is a linker that issusceptible to fragmentation on collision with another molecule or atomand X is preferably a pre-ionised, fragmentation resistant group. Thesum of the masses of M and X is the same for all members of the set.Preferably M and X have the same basic structure or core structure, thisstructure being modified by the mass adjuster moieties.

The mass adjuster moiety ensures that the sum of the masses of M and Xin is the same for all mass labels in a set, but ensures that each X hasa distinct (unique) mass.

A preferred set of mass labels having the above structure is one whereineach of the labels in the set has the following structure:

wherein R is hydrogen or is a substituted or unsubstituted aliphatic,aromatic, cyclic or heterocyclic group, L is the cleavable linker and Ais the mass adjuster moiety, each p is the same and is an integer of 0or greater, each y′ may be the same or different and is an integer of0-4, the sum of all y′ being equal to y, each z′ may be the same ordifferent and is an integer of 0-4, the sum of all z′ being equal to z.Preferably R is H, L is an amide bond, p=0, and A is an F atom.

In the present context, the substitution pattern on the R group is notat all limited. The substituent or substituents may comprise any organicgroup and/or one or more atoms from any of groups IIIA, IVA, VA, VIA orVIIA of the Periodic Table, such as a B, Si, N, P, O, or S atom or ahalogen atom (e.g. F, Cl, Br or I).

When the substituent comprises an organic group, the organic group maycomprise a hydrocarbon group. The hydrocarbon group may comprise astraight chain, a branched chain or a cyclic group. Independently, thehydrocarbon group may comprise an aliphatic or an aromatic group. Alsoindependently, the hydrocarbon group may comprise a saturated orunsaturated group.

When the hydrocarbon comprises an unsaturated group, it may comprise oneor more alkene functionalities and/or one or more alkynefunctionalities. When the hydrocarbon comprises a straight or branchedchain group, it may comprise one or more primary, secondary and/ortertiary alkyl groups. When the hydrocarbon comprises a cyclic group itmay comprise an aromatic ring, an aliphatic ring, a heterocyclic group,and/or fused ring derivatives of these groups. The cyclic group may thuscomprise a benzene, naphthalene, anthracene, indene, fluorene, pyridine,quinoline, thiophene, benzothiophene, furan, benzofuran, pyrrole,indole, imidazole, thiazole, and/or an oxazole group, as well asregioisomers of the above groups.

The number of carbon atoms in the hydrocarbon group is not especiallylimited, but generally the hydrocarbon group comprises from 1-40 Catoms. The hydrocarbon group may thus be a lower hydrocarbon (1-6 Catoms) or a higher hydrocarbon (7 C atoms or more, e.g. 7-40 C atoms).The number of atoms in the ring of the cyclic group is not especiallylimited, but the ring of the cyclic group may comprise from 3-10 atoms,such as 3, 4, 5, 6 or 7 atoms.

The groups comprising heteroatoms defined above, as well as any of theother groups defined above, may comprise one or more heteroatoms fromany of groups IIIA, IVA, VA, VIA or VIIA of the Periodic Table, such asa B, Si, N, P, O, or S atom or a halogen atom (e.g. F, Cl, Br or I).Thus the substituent may comprise one or more of any of the commonfunctional groups in organic chemistry, such as hydroxy groups,carboxylic acid groups, ester groups, ether groups, aldehyde groups,ketone groups, amine groups, amide groups, imine groups, thiol groups,thioether groups, sulphate groups, sulphonic acid groups, and phosphategroups. The substituent may also comprise derivatives of these groups,such as carboxylic acid anhydrydes and carboxylic acid halides.

In addition, any substituent may comprise a combination of two or moreof the substituents and/or functional groups defined above.

The arrays of mass labels of the present invention are not particularlylimited, provided that they contain a plurality of sets of mass labelsaccording to the present invention. It is preferred that the arrayscomprise two or more, three or more, four or more, or five or more setsof mass labels. Preferably each mass label in the array has either ofthe following structures:(S)_(x)-M(A)_(y)-L-X(A)_(z)M(A)_(y)-(S)_(x)-L-x(A)_(z)wherein S is the mass series modifying group, M is the massnormalisation moiety, X is the mass marker moiety, A is the massadjuster moiety, L is the cleavable linker, x is an integer of 0 orgreater, y and z are integers of 0 or greater, and y+z is an integer of1 or greater.

A preferred array of mass labels of the above type is one in which themass labels have either of the following structures:

wherein R is hydrogen or is a substituted or unsubstituted aliphatic,aromatic, cyclic or heterocyclic group, each p is the same and is aninteger of 0 or greater, x is an integer of 0 or greater each x for anyone set being different from the x of every other set in the array, eachy′ may be the same or different and is an integer of 0-4, the sum of ally′ being equal to y, and each z′ may be the same or different and is aninteger of 0-4, the sum of all z′ being equal to z. An array of thistype is depicted in FIG. 7.

In an alternative preferred aspect, the array of mass labels maycomprise mass labels having either of the following structures:S(A*)_(r)-M(A)_(y)-L-X(A)_(z)M(A)_(y)-S(A*)_(r)L-X(A)_(z)wherein S is a mass series modifying group, M is the mass normalisationmoiety, X is the mass marker moiety, A is a mass adjuster moiety of themass marker and mass normalisation moieties, A* may be the same ordifferent from A and is a mass adjuster moiety of the mass seriesmodifying groups, L is the cleavable linker, r is an integer of 0 orgreater and is at least 1 for one or more sets of mass labels in thearray, y and z are integers of 0 or greater, and x+y is an integer of 1or greater. Preferably, M is a fragmentation resistant group, L is alinker that is susceptible to fragmentation on collision with anothermolecule or atom and X is preferably a pre-ionised, fragmentationresistant group. S is typically a group such that each member of thearray of sets of labels comprises an S whose mass is separated bypreferably 4 daltons from every other S of every other member of thearray. Thus each different set of mass labels has a distinct (unique)mass.

A preferred array of mass labels of the above latter type is one inwhich the mass labels in the array has either of the followingstructures:

wherein R is hydrogen or is a substituted or unsubstituted aliphatic,aromatic, cyclic or heterocyclic group, each p is the same and is aninteger of 0 or greater, x is an integer of 0 or greater x being thesame for all mass labels in the array, each y′ may be the same ordifferent and is an integer of 0-4, the sum of all y′ being equal to y,each z′ may be the same or different and is an integer of 0-4, the sumof all z′ being equal to z, and each r′ may be the same or different,the sum of all r′ being equal to r. An array of this type is depicted inFIG. 6.

In the above sets and arrays of this invention, the common basicstructure of the M, X and S groups is not particularly limited and maycomprise a cyclic and/or a non-cyclic group. The nature of M, X and S isnot particularly limited. However, it is preferred that M and/or X,and/or S comprise as a basic (core) structure, a cyclic group, such asan aryl, a cycloalkyl or a heterocyclic group. These groups may beunsubstituted, but are preferably substituted. M, X and/or S mayrespectively comprise an oligomer or polymer formed from the abovecyclic monomers, where the cyclic monomers are linked by a fragmentationresistant bond or group.

Aryl ethers, such as a phenyl ether group and their oligomers andpolymers, especially substituted aryl ethers, are preferred common basicstructures for M, X and S.

The cleavable linker group L is not particularly limited. However, it ispreferred that L comprises a group which is cleavable by collision,and/or is cleavable in a mass spectrometer. Preferably the group Lcomprises an amide bond.

In a further preferred aspect, this invention provides sets and arraysof mass labels which can be reacted with analyte molecules, the masslabels having the form:Re-L′-label or Re-L′-S-labelwhere Re is a reactive functionality or group which allows the masslabel to be reacted covalently to an appropriate functional group in ananalyte molecule, such as, but not limited to, a nucleotideoligonucleotide, polynucleotide, amino acid, peptide or polypeptide. L′is a linker which may or may not be cleavable, and label is a mass labelfrom any of the sets or arrays defined above. S has the same meaning asdefined above. L′ may be a cleavable linker if desired, such as acleavable linker L, as defined above.

In preferred embodiments of the above aspects of the invention, L and/orL′ are cleavable within the mass spectrometer and preferably within theion source of the mass spectrometer.

Linker Groups

In the discussion above and below reference is made to linker groupswhich may be used to connect molecules of interest to the mass labelcompounds of this invention. A variety of linkers is known in the artwhich may be introduced between the mass labels of this invention andtheir covalently attached analyte. Some of these linkers may becleavable. Oligo- or poly-ethylene glycols or their derivatives may beused as linkers, such as those disclosed in Maskos, U. & Southern, E. M.Nucleic Acids Research 20: 1679-1684, 1992. Succinic acid based linkersare also widely used, although these are less preferred for applicationsinvolving the labelling of oligonucleotides as they are generally baselabile and are thus incompatible with the base mediated de-protectionsteps used in a number of oligonucleotide synthesisers.

Propargylic alcohol is a bifunctional linker that provides a linkagethat is stable under the conditions of oligonucleotide synthesis and isa preferred linker for use with this invention in relation tooligonucleotide applications. Similarly 6-aminohexanol is a usefulbifunctional reagent to link appropriately funtionalised molecules andis also a preferred linker.

A variety of known cleavable linker groups may be used in conjunctionwith the compounds of this invention, such as photocleavable linkers.Ortho-nitrobenzyl groups are known as photocleavable linkers,particularly 2-nitrobenzyl esters and 2-nitrobenzylamines, which cleaveat the benzylamine bond. For a review on cleavable linkers seeLloyd-Williams et al., Tetrahedron 49, 11065-11133, 1993, which covers avariety of photocleavable and chemically cleavable linkers.

WO 00/02895 discloses the vinyl sulphone compounds as cleavable linkers,which are also applicable for use with this invention, particularly inapplications involving the labelling of polypeptides, peptides and aminoacids. The content of this application is incorporated by reference.

WO 00/02895 discloses the use of silicon compounds as linkers that arecleavable by base in the gas phase. These linkers are also applicablefor use with this invention, particularly in applications involving thelabelling of oligonucleotides. The content of this application isincorporated by reference.

In the discussion below, reference is made to reactive functionalities,Re, to allow compounds of the invention to be linked to other compounds,whether reporter groups or analyte molecules. A variety of reactivefunctionalities may be introduced into the mass labels of thisinvention.

Table 1 below lists some reactive functionalities that may be reactedwith nucleophilic functionalities which are found in biomolecules togenerate a covalent linkage between the two entities. For applicationsinvolving synthetic oligonucleotides, primary amines or thiols are oftenintroduced at the termini of the molecules to permit labelling. Any ofthe functionalities listed below could be introduced into the compoundsof this invention to permit the mass markers to be attached to amolecule of interest. A reactive functionality can be used to introducea further linker groups with a further reactive functionality if that isdesired. Table 1 is not intended to be exhaustive and the presentinvention is not limited to the use of only the listed functionalities.

TABLE 1 Nucleophilic Functionality Reactive Functionality ResultantLinking Group —SH —SO₂—CH═CR₂ —S—CR₂—CH₂—SO₂— —NH₂ —SO₂—CH═CR₂—N(CR₂—CH₂—SO₂—)₂ or —NH—CR₂—CH₂—SO₂— —NH₂

—CO—NH— —NH₂

—CO—NH— —NH₂ —NCO —NH—CO—NH— —NH₂ —NCS —NH—CS—NH— —NH₂ —CHO —CH₂—NH——NH₂ —SO₂Cl —SO₂—NH— —NH₂ —CH═CH— —NH—CH₂—CH₂— —OH —OP(NCH(CH₃)₂)₂—OP(═O)(O)O—

It should be noted that in applications involving labellingoligonucleotides with the mass markers of this invention, some of thereactive functionalities above or their resultant linking groups mighthave to be protected prior to introduction into an oligonucleotidesynthesiser. Preferably unprotected ester, thioether and thioesters,amine and amide bonds are to be avoided, as these are not usually stablein an oligonucleotide synthesiser. A wide variety of protective groupsis known in the art which can be used to protect linkages from unwantedside reactions.

In the discussion below reference is made to “charge carryingfunctionalities” and solubilising groups. These groups may be introducedinto the mass labels such as in the mass markers of the invention topromote ionisation and solubility. The choice of markers is dependent onwhether positive or negative ion detection is to be used. Table 2 belowlists some functionalities that may be introduced into mass markers topromote either positive or negative ionisation. The table is notintended as an exhaustive list, and the present invention is not limitedto the use of only the listed functionalities.

TABLE 2 Positive Ion Mode Negative Ion Mode —NH₂ —SO₃ ⁻ —NR₂ —PO₄ ⁻ —NR₃⁺ —PO₃ ⁻

—CO₂ ⁻

—SR₂ ⁺

WO 00/02893 discloses the use of metal-ion binding moieties such ascrown-ethers or porphyrins for the purpose of improving the ionisationof mass markers. These moieties are also be applicable for use with themass markers of this invention.

The components of the mass markers of this invention are preferablyfragmentation resistant so that the site of fragmentation of the markerscan be controlled by the introduction of a linkage that is easily brokenby Collision Induced Dissociation. Aryl ethers are an example of a classof fragmentation resistant compounds that may be used in this invention.These compounds are also chemically inert and thermally stable. WO99/32501 discusses the use of poly-ethers in mass spectrometry ingreater detail and the content of this application is incorporated byreference.

In the past, the general method for the synthesis of aryl ethers wasbased on the Ullmann coupling of arylbromides with phenols in thepresence of copper powder at about 200° C. (representative reference: H.Stetter, G. Duve, Chemische Berichte 87 (1954) 1699). Milder methods forthe synthesis of aryl ethers have been developed using a different metalcatalyst but the reaction temperature is still between 100 and 120° C.(M. Iyoda, M. Sakaitani, H. Otsuka, M. Oda, Tetrahedron Letters 26(1985) 477). This is a preferred route for the production of poly-ethermass labels. See synthesis of FT77 given in the examples below. Arecently published method provides a most preferred route for thegeneration of poly-ether mass labels as it is carried out under muchmilder conditions than the earlier methods (D. E. Evans, J. L. Katz, T.R. West, Tetrahedron Lett. 39 (1998) 2937).

The present invention also provides a set of two or more probes, eachprobe in the set being different and being attached to a unique masslabel or a unique combination of mass labels, from a set or an array ofmass labels as defined as defined above.

Further provided is an array of probes comprising two or more sets ofprobes, wherein each probe in any one set is attached to a unique masslabel, or a unique combination of mass labels, from a set of mass labelsas defined above, and wherein the probes in any one set are attached tomass labels from the same set of mass labels, and each set of probes isattached to mass labels from unique sets of mass labels from an array ofmass labels as defined above.

In one embodiment, each probe is preferably attached to a uniquecombination of mass labels, each combination being distinguished by thepresence or absence of each mass label in the set of mass labels and/orthe quantity of each mass label attached to the probe. This is termedthe “mixing mode” of the present invention, since the probes may beattached to a mixture of mass labels.

In the above aspects, the nature of the probe is not particularlylimited. However, preferably each probe comprises a biomolecule. Anybiomolecule can be employed, but the biomolecule is preferably selectedfrom a DNA, an RNA, an oligonucleotide, a nucleic acid base, a peptide,a polypeptide, a protein and an amino acid.

In one preferred embodiment, this invention provides sets and arrays ofmass labelled analytes, such as nucleotides, oligonucleotides andpolynucleotides, of the form:Analyte-L′-label or Analyte-L′-S-label

Wherein L′ and S are as defined above, and label is a mass label fromany of the sets and arrays defined above.

In the above aspect, the nature of the analyte is not particularlylimited. However, preferably each analyte comprises a biomolecule. Anybiomolecule can be employed, but the biomolecule is preferably selectedfrom a DNA, an RNA, an oligonucleotide, a nucleic acid base, a peptide,a polypeptide, a protein and an amino acid.

In one embodiment, each analyte is preferably attached to a uniquecombination of mass labels, each combination being distinguished by thepresence or absence of each mass label in the set of mass labels and/orthe quantity of each mass label attached to the probe. As mentionedabove, this is termed the “mixing mode” of the present invention, sincethe probes may be attached to a mixture of mass labels.

As mentioned above, the present invention provides a method of analysis,which method comprises detecting an analyte by identifying by massspectrometry a mass label or a combination of mass labels unique to theanalyte, wherein the mass label is a mass label from a set or an arrayof mass labels as defined above. The type of method is not particularlylimited, provided that the method benefits from the use of the masslabels of the present invention to identify an analyte. The method maybe, for example, a method of sequencing nucleic acid or a method ofprofiling the expression of one or more genes by detecting quantities ofprotein in a sample. The method is especially advantageous, since it canbe used to readily analyse a plurality of analytes simultaneously.However, the method also has advantages for analysing single analytesindividually, since using the present mass labels, mass spectra whichare cleaner than conventional spectra are produced, making the methodaccurate and sensitive.

In a further preferred embodiment, the present invention provides amethod which method comprises:

-   -   (a) contacting one or more analytes with a set of probes, or an        array of probes, each probe in the set or array being specific        to at least one analyte, wherein the probes are as defined        above,    -   (b) identifying an analyte, by detecting the probe specific to        that analyte.

In this embodiment it is preferred that the mass label is cleaved fromthe probe prior to detecting the mass label by mass spectrometry.

The nature of the methods of this particular embodiment is notespecially limited. However, it is preferred that the method comprisescontacting one or more nucleic acids with a set of hybridisation probes.The set of hybridisation probes typically comprises a set of up to 2564-mers, each probe in the set having a different combination of nucleicacid bases. This method may be suitable for identifying the presence oftarget nucleic acids, or alternatively can be used in a stepwise methodof primer extension sequencing of one or more nucleic acid templates.

The mass labels of the present invention are particularly suitable foruse in methods of 2-dimensional analysis, primarily due to the largenumber of labels that can be simultaneously distinguished. The labelsmay thus be used in a method of 2-dimensional gel electrophoresis, or ina method of 2-dimensional mass spectrometry.

Thus, in one aspect the present invention provides a method of2-dimensional mass spectrometric analysis, which method comprises;

-   -   (a) providing one or more analytes, each analyte being labelled        with a mass label or a combination of mass labels unique to that        analyte, wherein the mass labels are from a set or array of mass        labels as defined above; e    -   (b) cleaving the mass labels from the analytes;    -   (c) detecting the mass labels;    -   (d) dissociating the mass labels in the mass spectrometer, to        release the mass marker moieties from the mass normalisation        moieties;    -   (e) detecting the mass marker moieties; and    -   (f) identifying the analytes on the basis of the mass spectrum        of the mass labels in the first dimension and the mass spectrum        of the mass marker moieties in the second dimension.

In this method, preferably in step (c) mass labels of a chosen mass or achosen range of masses are selected for detection. It is also preferredthat in step (e) mass marker moieties having a specific mass or aspecific range of masses are selected for detection.

In another aspect, the present invention provides a method of analysis,which method comprises:

-   -   (a) subjecting a mixture of labelled analytes to a first        separation treatment on the basis of a first property of the        analytes;    -   (b) subjecting the resulting separated analytes to a second        separation treatment on the basis of a second property of the        analytes; and    -   (c) detecting an analyte by detecting its label;        wherein the analytes are labelled with a mass label from a set        or an array of mass labels as defined above.

The property of the analytes is not particularly limited. However, inthis embodiment in step (a) and/or step (b) the analytes are preferablyseparated according to their length or mass. It is further preferredthat in step (a) and/or step (b) the analytes are separated according totheir iso-electric point. Typically, the analytes comprise one or moreproteins, polypeptides, peptides, amino acids or nucleic acids, orfragments thereof. It is particularly preferred that gel electrophoresisis employed in each of the separation steps. In this embodiment, themethod is a method of 2-dimensional gel electrophoresis.

In a further aspect, the present invention provides a method forcharacterising nucleic acid, which comprises:

-   -   (a) providing a population of nucleic acid fragments, each        fragment having cleavably attached thereto a mass label from a        set or an array of mass labels as defined above for identifying        a feature of that fragment;    -   (b) separating the fragments on the basis of their length;    -   (c) cleaving each fragment to release its mass label; and    -   (d) determining each mass label by mass spectroscopy to relate        the feature of each fragment to the length of the fragment.

Typically, the method of this aspect of the invention is used forcharacterising cDNA. Preferably, this method comprises:

-   -   (a) exposing a sample comprising a population of one or more        cDNAs or fragments thereof to a cleavage agent which recognises        a predetermined sequence and cuts at a reference site at a known        displacement from the predetermined sequence proximal to an end        of each cDNA or fragment thereof so as to generate a population        of terminal fragments;    -   (b) ligating to each reference site an adaptor oligonucleotide        which comprises a recognition site for a sampling cleavage        agent;    -   (c) exposing the population of terminal fragments to a sampling        cleavage agent which binds to the recognition site and cuts at a        sampling site of known displacement from the recognition site so        as to generate in each terminal fragment a sticky end sequence        of a predetermined length of up to 6 bases, and of unknown        sequence;    -   (d) separating the population of terminal fragments into        sub-populations according to sequence length; and    -   (e) determining each sticky end sequence by:        -   (i) probing with an array of labelled hybridisation probes,            the array containing all possible base sequences of the            predetermined length;        -   (ii) ligating those probes which hybridised to the sticky            end sequences and        -   (iii) determining which probes are ligated by identification            and preferably quantification of the labels;            wherein the labels are mass labels from a set or an array as            defined above.

In this method, the population of terminal fragments is preferablyseparated by capillary electrophoresis, HPLC or gel electrophoresis.

In a still further aspect of the present invention, there is provided amethod for characterising nucleic acid, which method comprisesgenerating Sanger ladder nucleic acid fragments from one or more nucleicacid templates, in the presence of at least one labelled terminatingbase, and identifying the length of the fragment, and the terminatingbase of the fragment, wherein the label is specific to the terminatingbase and is a mass label from a set or an array as defined above.

In this aspect of the invention, it is preferred that all fourterminating bases are present in the same reaction zone. The methodtypically comprises generating Sanger ladder nucleic acid fragments froma plurality of nucleic acid templates present in the same reaction zone,and for each nucleic acid fragment produced identifying the length ofthe fragment, the identity of the template from which the fragment isderived and the terminating base of the fragment, wherein prior togenerating the fragments, a labelled primer nucleotide oroligonucleotide is hybridised to each template, the label on each primerbeing specific to the template to which that primer hybridises to allowidentification of the template. The type of label identifying thetemplate is not particularly limited. However, it is preferred that thelabel identifying the template is a mass label from a set or an array asdefined in above.

A further aspect of the method of the present invention provides amethod for sequencing nucleic acid, which method comprises:

-   -   (a) obtaining a target nucleic acid population comprising one or        more single-stranded DNAs to be sequenced, each of which is        present in a unique amount and bears a primer to provide a        double-stranded portion of the nucleic acid for ligation        thereto;    -   (b) contacting the nucleic acid population with an array of        hybridisation probes, each probe comprising a label cleavably        attached to a known base sequence of predetermined length, the        array containing all possible base sequences of that        predetermined length and the base sequences being incapable of        ligation to each other, wherein the contacting is carried out in        the presence of ligase under conditions to ligate to the        double-stranded portion of each nucleic acid the probe bearing        the base sequence complementary to the single-stranded nucleic        acid adjacent the double-stranded portion thereby to form an        extended double-stranded portion which is incapable of ligation        to further probes; and    -   (c) removing all unligated probes; followed by the steps of:    -   (d) cleaving the ligated probes to release each label;    -   (e) recording the quantity of each label; and    -   (f) activating the extended double-stranded portion to enable        ligation thereto;        wherein    -   (g) steps (b) to (f) are repeated in a cycle for a sufficient        number of times to determine the sequence of the or each        single-stranded nucleic acid by determining the sequence of        release of each label,        wherein the labels of the hybridisation probes are each from a        set or an array as defined above.

In this aspect of the invention, it is preferred that the hybridisationprobes are a set of 256 4-mers, each probe in the set having a differentcombination of nucleic acid bases.

As already mentioned, it is preferred in all of the above aspects of thepresent methods that two or more analytes are detected by simultaneouslyidentifying their mass labels or combinations of mass labels by massspectrometry.

The mixing mode of the present invention may be applied to all of theabove methods. In this embodiment, each analyte is identified by aunique combination of mass labels from a set or array of mass labels,each combination being distinguished by the presence and absence of eachmass label in the set or array and/or the quantity of each mass label.

If the method is applied to two or more analytes simultaneously, in someaspects it is preferred that the analytes are separated according totheir mass, prior to detecting the mass label by mass spectrometry.Preferably, the separation step is a chromatographic step, such asliquid chromatography or gel electrophoresis. The present labels of type2 are particularly advantageous in these embodiments, since theaggregate mass of all labels in the set is the same, thus during achromatographic separation step, the mobility of all analytes is equallyaffected by the labels.

Typically, in the present methods, the mass spectrometer employed todetect the mass label comprises one or more mass analysers, which massanalysers are capable of allowing ions of a particular mass, or range ofmasses, to pass through for detection and/or are capable of causing ionsto dissociate. Preferably ions of a particular mass or range of massesspecific to one or more known mass labels are selected using the massanalyser, the selected ions are dissociated, and the dissociationproducts are detected to identify ion patterns indicative of theselected mass labels. In particularly preferred methods, the massspectrometer comprises three quadrupole mass analysers. In thisembodiment, generally a first mass analyser is used to select ions of aparticular mass or mass range, a second mass analyser is used todissociate the selected ions, and a third mass analyser is used todetect resulting ions.

A preferred embodiment of the above methods provides a method ofanalysing mass labelled analyte molecules, comprising the steps of:

-   -   1. Cleaving the mass label from its associated molecule of        interest.    -   2. Ionising the cleaved mass label.    -   3. Selecting ions of a predetermined mass to charge ratio        corresponding to the mass to charge ratio of the preferred ions        of known mass labels in a mass analyser.    -   4. Inducing dissociation of these selected ions by collision.    -   5. Detecting the collision products to identify collision        product ions that are indicative of the selected mass labels.

It is preferred that the process of cleaving the mass label from itsassociated nucleic acid takes place within a mass spectrometer,preferably within the ion source. It is also preferred that the masslabels are pre-ionised. In this embodiment the labels need only betransferred from a liquid or solid phase into the gas phase (if the masslabels are in a liquid or solid phase). Typically, the step of ionisingthe mass label results from cleavage of the mass label within the ionsource of the mass spectrometer.

Preferably, the third step of selecting the ions of a predetermined massto charge ratio is performed in the first mass analyser of a serialinstrument. The selected ions are then channelled into a separatecollision cell where they are collided with a gas or a solid surfaceaccording to the above fourth step. The collision products are thenchannelled into a further mass analyser of a serial instrument to detectcollision products according to the above fifth step. Typical serialinstruments for use in the present invention include triple quadrupolemass spectrometers, tandem sector instruments and quadrupole time offlight mass spectrometers.

It is further preferred that the above third step of selecting the ionsof a predetermined mass to charge ratio, the fourth step of collidingthe selected ions with a gas and the fifth step of detecting thecollision products are performed in the same zone of the massspectrometer. This may, for example, be effected in ion trap massanalysers and Fourier Transform Ion Cyclotron Resonance massspectrometers.

In a further preferred embodiment, the invention provides a method ofanalysing mass labelled analyte molecules, comprising the steps of:

-   -   1. Cleaving the mass label from its associated analyte molecule.    -   2. Ionising the cleaved mass label.    -   3. Selecting ions of a predetermined mass to charge ratio        corresponding to the mass to charge ratio of the preferred ions        of known mass labels in a mass analyser.    -   4. Inducing dissociation of these selected ions by collision.    -   5. Detecting more than one of the collision products to identify        collision product ion patterns that are indicative of the        selected mass labels which in turn identify the labelled nucleic        acid.

In preferred aspects of this embodiment of this invention, the processof cleaving the mass label from its associated nucleic acid takes placewithin a mass spectrometer, preferably within the ion source.

In certain preferred aspects of this embodiment, the mass labels arepre-ionised and need only be transferred from a liquid or solid phaseinto the gas phase (if the mass labels are in a liquid or solid phase).

In other preferred aspects, the step of ionising the mass label resultsfrom cleavage of the mass label within the ion source of the massspectrometer.

In certain aspects, the third step of selecting the ions of apredetermined mass to charge ratio is performed in the first massanalyser of a serial instrument. The selected ions are then channelledinto a separate collision cell where they are collided with a gas or asolid surface according to the above fourth step. The collision productsare then channelled into a further mass analyser of a serial instrumentto detect collision products according to the above fifth step. Typicalserial instruments include triple quadrupole mass spectrometers, tandemsector instruments and quadrupole time of flight mass spectrometers.

In other preferred aspects, the third step of selecting the ions of apredetermined mass to charge ratio, the fourth step of colliding theselected ions with a gas and the fifth step of detecting the collisionproducts are performed in the same zone of the mass spectrometer.

This may effected in ion trap mass analysers and Fourier Transform IonCyclotron Resonance mass spectrometers, for example.

Tandem Mass Spectrometry

At the expense of some loss in sensitivity, great gains in selectivitycan be gained through use of tandem mass spectrometry (MS/MS) to detectthe mass labels of the present invention. For the purposes ofillustrating the invention some discussion is now provided regardingtandem mass spectrometry, exemplified here by reference to the triplequadrupole mass spectrometer. The triple quad allows easy illustrationof the principle of MS/MS.

The quadrupole mass analyser is essentially a mass filter which can atany moment be set to allow ions of only a particular mass to chargeratio to pass through. A quadrupole comprises 4 parallel rod shapedelectrodes which form a channel. A direct current potential superimposedby a sinusoidal radio frequency potential is applied to the rodelectrodes. Ions entering into the channel formed by the parallel rodsfollow complex trajectories and for a particular DC potential and radiofrequency potential, only ions with a predetermined mass to charge ratiowill have a stable trajectory which will lead them through the channel.By changing the applied potentials the quadrupole can be made to scanacross a full range of mass to charge ratios up to about 4000.

A triple quad (Q) layout is shown in FIG. 1. Three separate quadrupolemass analysers are linked in series. The first quadrupole is referred tohereafter as Q1, similarly the second will be referred to as Q2 and thethird as Q3. Quadrupoles Q1 and Q3 are typically used in scanning modes.The speed of scanning is very high. Alternatively, Q1 or Q3 can be usedas “gates”, which allow through only selected ions. Quadrupole Q2 isused in a non-scanning mode, in which it acts as an ion focusing device.All ions pass through Q2 when there is a high vacuum. When a gas isintroduced into Q2, incoming ions collide with gas and many of the ionsgain sufficient energy to fragment. This is “Collision InducedDissociation” (CID).

Consider one particular use of the triple quad. Suppose ions areproduced in the ion source (A⁺ B⁺, C⁺, D⁺, etc.). If all of these ionsare allowed through Q1, with Q2 and Q3 operating in a scanning mode,then a full mass spectrum is generated (FIG. 11—Mass Spectrum 1). MassSpectrum 1 shows a spectrum comprising the molecular ions A⁺ through toD⁺ and assorted fragment ions.

Now suppose Q1 is set to pass only A⁺ ions and Q2 is at low pressure.The A⁺ ions pass through Q2 and Q3 and are detected (FIG. 12—MassSpectrum 2). The new mass spectrum is now “cleaned”, of the other ions(B⁺ C⁺, etc.) having been rejected by Q1. Multiple analytes can bedetected from the same sample introduced into the mass spectrometer bysetting Q1 to scan over a limited series of mass corresponding toparticular ion species of the analytes of interest. This is termed“Selective Ion Monitoring”.

A triple quadrupole can be used to gain further selectivity, though. Itis possible that A⁺ ions may come from several sources (e.g. severalions could have the same mass to charge ratio of 100 but have differentcompositions such as C₇H₁₆, C₆H₁₂O, C₅H₈O etc.). Suppose there are twocompositions of A⁺ ions (A_(l) ⁺, A₂ ⁺) both of the same nominal mass(FIGS. 13 and 14—Mass Spectra 3 and 4). If A⁺ ions are selected in Q1and CID is carried out in Q2, a scan of Q3 will give the spectrum shownin FIG. 15—Mass Spectrum 5. This is a “mixed” spectrum.

Suppose it is known that ions P⁺, Q₊ (or even just P⁺) can unambiguouslyreveal that A₁ ⁺ is present. That is to say, the fragmentation(reaction) A₁ ⁺→P⁺+Q⁺ is known to occur. Instead of scanning all ions inQ3, it is set to detect only P⁺ ions. Thus, after leaving the ionsource, ions A⁺ B⁺ . . . are reduced to just A⁺ (=A₁ ⁺, A₂ ⁺) ions goinginto Q2.

After CID, only fragment ions P⁺ are selected and these arecharacteristic of only A₁ ⁺. This is said to be “Single or SelectiveReaction Monitoring”, which is highly selective. In a more generalisedsense, the full spectrum of ions entering Q1 (FIG. 11—Mass Spectrum 1)is reduced in Q3 to P⁺ (FIG. 16—Mass Spectrum 6) and these ions areknown to relate only to A₁ ⁺.

In some of the ensuing discussions, the examples refer to the use of themass labels of this invention to identify nucleotides oroligonucleotides. It is equally possible that the labels of thisinvention can be used with proteins or peptides or other analytes andoligonucleotides are mentioned for the purpose of example. For thepurposes of analysing oligonucleotides it is assumed that the masslabels are attached to the oligonucleotide covalently via a cleavablelinker. The linker may be cleavable by a variety of mechanisms,including thermal cleavage, chemical cleavage, cone voltage cleavage orphoto-cleavage. In the following discussion of the behaviour of masslabels it is assumed that the labels have been cleaved from theirassociated nucleic acids during or prior to ionisation. Preferredcleavable linkers and their methods of use are disclosed in GB patentapplications GB 9815163.2 and GB 9815164.0. The preferred cleavageprocess is represented schematically in FIG. 20.

According to the first aspect of this invention the principle ofSelected Ion Monitoring (SIM) coupled to Selected Reaction Monitoring(SRM) can be applied to mass marking techniques giving a 2-dimensionaldetection process. If A₁ ⁺ was an ion from a mass label and therefore ofknown composition and fragmentation pattern then no matter how many ionswere produced in the ionisation step, the mass label could be identifiedwithout there being any interference from other ions by gating A⁺ ionsin the first quadrupole of a triple quad and then detecting A₁ ⁺fragmentation products, i.e. by gating only P⁺ ions in the thirdquadrupole of a triple quadrupole. It would not matter which M/Z rangewas examined and it is no longer necessary to find “clean” windows inthe mass spectrum.

As mentioned above, one aspect of this invention provides mass labelswhich can be represented schematically by the formula M-L-X. As anexample A₁ ⁺ could be the molecular ion for the label shown below:

M is thus a benzyl group, L is an amide bond and X is a pyridyl group.The amide bond linking the benzyl ring to the pyridyl ring isparticularly susceptible to cleavage by collision. Thus, on collision,A₁ ⁺ produces the fragment ion below:

and this would represent P⁺. Thus, detection of P⁺ means A₁ ⁺ is presentand that one of the labels is present. The label has been selectivelyidentified from all other ions and this effectively eliminates“background” contamination. This means that labelled-analytes do notneed to be exhaustively purified and that the labels do not need to becleaved and separated from the analyte outside the mass spectrometer.This principle can be generalised to provide a useful class of compoundsfor use as mass labels, all of which have a general structure M-L-Xwhere M is connected to X via a scissile bond L, such as an amide bondand X is the ion that is detected by SRM. Thus X is analogous to thecleavage product shown above and referred to as P⁺.

According to one aspect of this invention the mass label structureillustrated above can be generalised to provide a useful set of masslabels all with the same mass but which are still easily resolved bySRM. Let M₀, M₁ . . . M₄ and X₀, X₁ . . . X₄ be isotopic forms of thehalves of M-L-X where L is an amide bond linking M and X. The exampleabove can be used again. If this structure is substituted with fluorine,the components shown in FIG. 2 can be generated. These label componentscan be combined to form a mass label MX (ignoring the cleavable bond Lfor the moment) as follows:M₀X₄; M₁X₃; M₂X₂; M₃X₁;M₄X₀

These five substances have exactly the same mass (FIG. 3). Thus, if amass label was selected in Q1 of a triple quadrupole, only ions ofmass=M_(m)X_(n) (m=0-4, n=4-0) would be selected. Q1 could be set to“look” for only MX ions. If CID is effected in Q2, then Q3 could be setto pass only ions X₀, X₁, X₂, X₃ and X₄ as shown in FIG. 21.

Therefore, all at the same mass, there would be 5 mass labels selectedin Q1 and the cleavage reactions shown below can be identified in Q3. Ifmass 139 were detected in Q3, it must have come from M₃X₁ and so on.M₀X₄ ⁺→X₄ ⁺ (m/z 193)M₁X₃ ⁺→X₃ ⁺ (m/z 175)M₂X₂ ⁺→X₂ ⁺ (m/z 157)M₃X₁ ⁺→X₁ ⁺ (m/z 139)M₀X₄ ⁺→X₀ ⁺ (m/z 121)

The selection process of this method can be visualised as atwo-dimensional mass spectrum shown in FIG. 17—mass spectrum 7.

In an alternative approach, a different set of mass labels can besynthesised. In this mode of analysis SRM is combined with “Selected IonMonitoring” (SIM). In the SIM mode of analysis, the first quadrupole(Q1) selectively scans over predetermined masses gating only ions withthe predetermined masses.

Considering M₀, M₁, M₂, M₃, M₄ and X₀ from FIGS. 2 and 3 again, theselabel components can be combined to give 5 labels with different masses,M₀X₀, M₁X₀, M₂X₀, M₃X₀ and M₄X₀. Now suppose Q1 of a triple quadrupoleis set to select these 5 masses, then Q3 need only be set to detect 1mass (X₀) as in FIG. 4.

Thus, the mass spectrometer identifies only 1 fixed ion (X₀). Since X₀must come from M₀X₀, M₁X₀, M₂X₀, M₃X₀, M₄X₀ only and it is known whenthese have been selected in Q1 then this provides an alternative mode ofmass marking. Five different analytes can now be identified by one offive specific “single reactions”M₀X₀→X₀M₁X₀→X₀M₂X₀→X₀M₃X₀→X₀M₄X₀→X₀

This generates a different 2-dimensional mass spectrum shown in FIG.18—mass spectrum 8.

The two approaches above can be combined. Suppose M₀, M₁, M₂, M₃ arechosen to represent the first base of a dinucleotide. The second base ischaracterised by X₀, X₁, X₂, X₃ to give 16 different mass labels asshown in Table 3 below:

TABLE 3 Dinucleotide AA AC AG AT Mass Label M₀X₃ M₀X₂ M₀X₁ M₀X₀Dinucleotide CC CA CG CT Mass Label M₁X₂ M₁X₃ M₁X₁ M₁X₀ Dinucleotide GGGA GC GT Mass Label M₂X₁ M₂X₃ M₂X₂ M₂X₀ Dinucleotide TT TA TC TG MassLabel M₃X₀ M₃X₃ M₃X₂ M₃X₁

Each mass label will have one of 7 different masses which can beselected in the first mass analyser of a tandem instrument. Thecollision products identified in the second mass analyser will identifythe dimer. Thus with 8 mass label components, it is possible to generate16 mass labels. The full mass 2-dimensional mass spectrum for all ofthese labels is shown in FIG. 19—mass spectrum 9. Similarly, if 256 masslabels are required, two sets of 16 components, i.e. M₀ to M₁₅ and X₀ toX₁₅, would generate sufficient labels, where each label would have oneof 31 different masses.

According to another aspect of this invention, it is also possible togenerate arrays of sets of mass labels using mass series modifyinggroups. According to this aspect of the invention a set of labels, whereeach label in the set has the same mass but can be resolved by SRM, canbe expanded into an additional set of labels by linking each member ofthe set to a mass series modifying group which will shift the mass ofeach member of the set by a pre-determined amount thus generating asecond set of labels whose total mass is different from the first set.Thus two distinct sets of mass label ions would be gated by SIM in thefirst quadrupole of a mass analyser and the collision products wouldthen be analysed in the third quad by monitoring the same fragmentspecies for both sets of labels. Clearly as many different sets oflabels as can be comfortably analysed in a mass spectrometer can begenerated by using different mass series modifying groups.

Mass series modifying (S) groups are preferably fragmentation resistantgroups such that each S group, when linked to each member of a set oflabels, generates a new set of labels that is clearly resolvable fromevery other in an array of such labels. In this context resolvable meansthat each set of labels in the array is preferably separated from everyother set by approximately 4 daltons at least. This is to ensure thatisotope peaks from one label do not overlap in the mass spectrum withthose of another label. In preferred embodiments of this aspect of theinvention, the S groups are substituted or unsubstituted cyclic groups,such as aryl groups, cycloalkyl groups and heterocyclic groups,preferably linked to the members of a set of SRM resolvable mass markinggroups by an ether linkage. Each set in the array may have the same Sgroup, but having a different level of substitution, to ensure that eachset is distinct from all other sets. An example of an array of suchlabels is shown in FIG. 6. In this array, F atoms are used assubstituents (adjuster moieties), but other substituents such as methylgroups could be employed. It should be clear that an array of suchlabels will have very similar effects on the mobility of any associatedanalyte molecules.

Additional sets of labels could be added to such an array using methylsubstituted phenyl groups and also phenyl groups substituted with bothmethyl and fluoro groups. Methyl groups differ in mass from fluorogroups by just less than 4 daltons and so a significant array of labelscould be generated whose effect on the mobility of associated analyteswould be minimal.

In other preferred embodiments of this aspect of this invention, the Sgroups are oligomers or polymers of cyclic groups such as aryl groups,cycloalkyl groups and heterocyclic groups, which may also besubstituted. Specifically, preferred S groups are poly-aryl ethers. Anexample of such an array is shown in FIG. 7.

According to a further aspect of this invention the principles describedabove can be taken further, by labelling analytes with a distinctcombination of the mass labels of the present invention. As mentionedabove, this embodiment is termed mixing mode labelling. When anyindividual analyte of a large number must be identified, for example incombinatorial chemistry, a mixture of labels, e.g. M₀X₃, M₁X₂, M₂X₁,M₃X₀ is chosen. The mixture is attached to an analyte, such that aparticular quantity of each label is present. For instance,aM₀X₃+bM₁X₂+cM₂X₁+dM₃X₀ where a=b=c=d=1 (FIG. 5). If equal parts of fourmass labels (a=b=c=d=0.25) are coupled to an analyte in the samereaction, the chemical joining reaction would not discriminate betweenthem. When an oligonucleotide is labelled, the oligo is mass marked withmore than one label per nucleotide or oligonucleotide.

Consider three mass labels of the form shown below in Table 4:

TABLE 4 Collision Product Mass Total (mass marker Name Structure Massmoiety) P

199 109 Q

199 108 R

199 107where “*” can represent ²H or ¹³C isotopes at the position marked. Itshould be clear that different substituents can be used such as fluorineor methyl groups for example. One mixing mode is such as that shown inFIG. 8. Eight distinct patterns can be generated by a combination of thepresence or absence of a distinct labels in a mixture coupled to ananalyte molecule.

Consider a different sort of pattern where the ratios of each of fivelabels are varied when they are coupled to their associated analyte asshown in Table 5 below:

TABLE 5 P Q R S T 2 2 2 2 2 2 2 2 1 2 2 2 2 0 2 2 2 1 2 2 . . . . . . .. . . . . . . . 0 0 1 0 2 0 0 0 2 2 0 0 0 1 2 0 0 0 0 2

With 4 mass labels, P, Q, R and S, which can be present at 3 differentratios, i.e. none, 1 or 2, there are effectively 3 different entitiesfor each label which means that there are a possible 81 different massspectral patterns that can be generated. It is preferable that there isalso one if these labels whose ratio to the other components remainsconstant (T), to act as an internal label against which the massspectrometer data system can compare the relative ratios of P, Q, R andS. This means that with a mixture of 5 labels all 64 combinations ofnatural nucleotides in a 3-mer oligonucleotide could be identified.

In the above example P, Q, R, S and T can be labels of the form shown inFIG. 3, thus the five labels have the same mass and can be gated frombackground contaminants in the first quadrupole of a triple quadruple ora Q-TOF instrument, for example. The fragmentation patterns formed as aresult of collision with a bath gas in the second quadrupole of a triplequadrupole and detection in the third quadrupole are shown in FIG. 9.

It may be seen that the principle can be extended. In some aspects ofthe present invention it is desirable to label the 256 possible 4-mers,using the above strategy. It is necessary to generate 7 different labelswhich can be mixed in all of the possible combinations of ratios shownabove. Alternatively, if the labels are of the form shown in FIG. 6 or7, then 4 sets of 81 codings of the sort shown in the example above canbe generated by using the 4 different mass series modifying groups togenerate different sets of 5 labels as shown in FIG. 6. This generatessufficient labels to encode all possible 256 4s.

The principle of this aspect of the invention can be extended stillfurther. Consider a library of DNA 4-mers comprising all 256 possiblecombinations of the natural nucleotides. Each 4-mer in the series can berepresented as a number from 1 to 256, i.e. AAAA would be 1, and AAACwould be 2 through to TTTT which would be 256.

The numbers 1 to 256 can be represented in a binary form for example inthe way numbers could be represented in a memory register of a computer.In a register there is a series of switches which represent the numbers2⁸, 2⁷, 2⁶, 2⁵, 2⁴, 2³, 2², 2¹ and 2⁰. To represent any of the numbersfrom 1 to 256, the switches are turned on and off so that the sum of thebinary powers represents the original decimal number, as shown in Table6 below:

TABLE 6 2⁸ 2⁷ 2⁶ 2⁵ 2⁴ 2³ 2² 2¹ 2⁰  1 Off Off Off Off Off Off Off OffOff  2 Off Off Off Off Off Off Off On Off  3 Off Off Off Off Off Off OffOn On  4 Off Off Off Off Off Off On Off Off . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 255 Off On On On On On On On On 256 OnOff Off Off Off Off Off Off Off

An analogous representation of these numbers can be achieved with masslabel molecules where each switch in the register is represented by thepresence or absence of a particular molecule. Thus to identify a 4-merone could label the 4-mer with the mixture of labels that represents thenumber that identifies that 4-mer, e.g. if AACG is represented by thenumber 7, it can be identified by labelling the 4-mer with a mixture ofa molecule that represents 2² with molecules that represent 2¹ and 2⁰.

Practically speaking, these molecules can be represented as a series ofmolecules based on a core molecule substituted with different numbers ofa particular substituent or isotope, e.g. different numbers of a massadjuster moiety, such as fluorine atoms or different deuterium isotopes.Thus 2⁰ can be represented by the core molecule with no fluorinesubstituents, 2¹ can be represented by the core molecule with 1 fluorinesubstituent and similarly 2⁸ can be represented by the core moleculewith 8 fluorine substituents. When these molecules are analysed by massspectrometry, they can be combined with a complementary component togive 9 isobaric tags which can be analysed in a tandem instrument.

Thus, in the case of the 4-mer AACG, this oligo can be labelled withlabels 0, 1 and 2 from the labels shown above. Clearly all otherpossible 4-mers can be represented in this binary fashion and requireonly 8 basic labels to identify them such as shown in FIG.

DNA Sequencing Using SRM

The analysis of Sanger Sequencing Ladders can be effected efficientlyusing mass labels of the form discussed above. Conventional DNAsequencing according to the Sanger methodology uses a DNA polymerase toadd numerous dideoxy/deoxynucleotides to an oligonucleotide primer,annealed to a single stranded DNA template, in a template specificmanner. Random termination of this process is achieved when terminatingnucleotides, i.e. the dideoxynucleotides, are incorporated into thetemplate complement. A “DNA ladder” is produced when the randomlyterminated strands are separated on a denaturing polyacrylamide gel orin a capillary. Sequence information is gathered, generally usingpolyacrylamide gel electrophoresis to separate the terminated fragmentsby length, followed by detecting the “DNA ladder”. In conventionalsemi-automated and automated DNA sequencers, such as the ABI377 fromPerkin Elmer or MegaBACE from Molecular Dynamics, fluorescent labels F₁,F₂, F₃, F₄ are used to identify the four terminating bases A, C, G, T.either through incorporating the fluorescent label into one of theterminating nucleotides or the primer used in the reaction. This ladderis then read by looking for the four dyes passing a detector which scansthe gel or a capillary. Other fluorescent detection formats arepossible.

Sequencing a Single Template

In the mass spectral method, the fluorescent labels are exchanged formass labels (e.g. M₀X₄; M₁X₃; M₂X₂; M₃X₁; M₄X₀ shown in FIG. 3). Thedideoxy terminator for Adenine is now labelled with M₁X₃. Similarly thedideoxy terminator for Cytosine is now labelled with M₂X₂, theterminator for Guanosine is labelled with M₃X₁ and the terminator forThymidine is labelled with M₄X₀. As the bands elute from the capillary,they are sprayed, in-line, into the ion source of a suitable tandem massanalyser such as a triple quadrupole where the mass marked nucleic acidsare analysed according to an aspect of this invention. Typically, in theion source the labels are cleaved from the terminating base of eachfragment in the ladder and enter the first mass analyser, Q1 of a triplequadrupole. Q1 is set to gate only molecular ions of MX, while Q3 is setto look for labels X₀ through X₄. It may be desirable that one of themasses, say X₄, should be used as an internal standard, viz.; it isalways present and X₀, X₁, X₂, and X₃ are examined in relation to X₄.

In an alternative approach, the four terminating nucleotides can belabelled with the four labels shown in FIG. 4 so that the dideoxyterminator for Adenine is now labelled with M₁X₀. Similarly the dideoxyterminator for Cytosine is now labelled with M₂X₀, the terminator forGuanosine is labelled with M₃X₀ and the terminator for Thymidine islabelled with M₄X₀. The label M₀X₀ can be used as an internal standardif desired. In this embodiment, Q1 of a triple quadrupole is set to gatemolecular ions of labels M₄X₀ through M₀X₀, while Q3 is set to look forlabels X₀.

In addition to nucleotide terminator labelled sequencing, primerlabelled sequencing can be performed. Details of primer labelledsequencing, which can employ the mass labels of the present inventionare provided in PCT/GB98/02048.

Multiplexed Sequencing of Templates with Mass Labels

The mass labels of this invention permit more than one template to beanalysed simultaneously, since many more than four labels can bedeveloped. This means that multiple sets of four labels can be generatedto permit analysis of multiple templates according to the methodologydescribed above which is based on the methods devised originally bySanger. Details regarding the multiplexed sequencing of nucleic acidtemplates which can employ the mass labels of the present invention areprovided in PCT/GB98/02048.

Mass labels of the form shown in FIG. 6 or FIG. 7 can be used tomultiplex the analysis of multiple DNA sequences. Each set of fivelabels, resolvable in the mass spectrometer from every other set by adistinct mass series modifier, can be used to identify a singletemplate, with a spare label remaining for use as a size/quantitystandard if desired. However, sets of 4 labels are sufficient forsequencing, and size standards are not essential. It is thus possible,for example, to use the array of 20 labels shown in FIG. 6 to analysethe Sanger reaction products of 5 templates simultaneously.

Gene Expression Profiling

Various methods of analysing populations of complementary DNA derivedfrom poly-adenylated messenger RNA have been developed. A number ofthese methods are based on detecting different sized amplification orrestriction products by electrophoretic separations of amplified cDNAlibraries. In general these techniques are based on generatingcharacteristic restriction fragments or amplification products from themembers of a complementary DNA (cDNA) library derived frompoly-adenylated messenger RNA.

Differential Display (Laing and Pardee, Science 257, 967-971, 1992) isthe classical method of electrophoresis based gene expression profiling.Developments of the concepts of this technique have been made resultingin improved successors to this technique. Expression profiling methodsbased on “molecular indexing” using type IIS or type IP restrictionendonucleases such as Sibson (PCT/GB93/0145) or Kato (EP 0 735 144 A1)are examples of one class of successors. In particular WO 98/48047discloses a molecular indexing method based on capillary electrophoresismass spectrometry (CEMS).

In this method cDNAs are synthesised using anchored and biotinylatedpoly-thymidine primers, which ensure that all cDNAs are terminated witha short poly-A tail of fixed length. In an “anchored primer” cDNApreparation, poly-A carrying mRNAs are captured and primed using anoligonucleotide of about 18 deoxythymidine residues with one of thethree remaining bases at the 3′ end to anchor the primer at the end ofthe poly-A tract. Biotinylation of the primers allows the cDNAs to beimmobilised on an avidinated solid phase support. These captured cDNAsmay be cleaved with an ordinary type II restriction endonuclease. Thisleaves a 3′ terminal restriction fragment on the solid support whileother fragments are washed away. An adapter is ligated to the resultingknown sticky-end. The adapter is designed to carry the binding site fora type IIs restriction endonuclease. These enzymes bind their targetsequence but cleave the underlying DNA at a defined number of bases awayfrom the binding site. Certain of these enzymes produce a staggered cut;fok1 for example will generate an ambiguous 4 bp sticky-end. If apopulation of cDNAs is treated with such an enzyme the sticky end willbe exposed at the adaptered terminus of each cDNA in the population. Afamily of adapter molecules is used to probe those 4 exposed bases. Witha 4 bp ambiguous sticky-end there are 256 possible candidates. Toidentify the probes, they are tagged with mass labels using a cleavablelinker, so that a unique mass label identifies each of the 256 possible4 bp adapters. This results in a population of fragments with varyinglengths according to where the ordinary type II restriction endonucleasecut them and with one of 256 possible mass labelled adapters at the 5′terminus of the cDNA.

The mass labelled 3′ restriction fragments are then separated on thebasis of their length, using capillary electrophoresis, followed byanalysis of the mass labels ligated to the termini of the cDNAfragments. The CE column feeds directly into an electrospray massspectrometer or equivalent mass spectrometer. On ionisation in the massspectrometer the labels cleave from their associated restrictionfragments. The quantity of each mass label present in each band,corresponding to a different restriction fragment length, eluting fromthe capillary electrophoresis column is determined. This process gives asignature for each cDNA that can be used to search a database.

This technique preferably uses 256 mass labels. Using conventionalapproaches to mass labelling would result in an array of mass tags,separated by about 4 daltons, spanning a mass range of more than athousand daltons. It is unlikely that an array of such labels could begenerated where all the tags had the same effect on the mobility of theassociated cDNA restriction fragments. This would mean that complexcorrection algorithms would have to be used to account for differencesin mobility and allow accurate determination of fragment length. Themass markers and associated mass labels of this invention, however, areeminently suitable in the above method, for generating arrays of massmarkers whose effect on the mobility of associated analyte molecules isthe same allowing direct determination of fragment length with highsensitivity and excellent signal to noise ratios.

A second class of electrophoretic techniques is based on the use ofordinary type II restriction endonucleases, which are used to introduceprimer sequences into cDNA restriction fragments. PCR amplification withlabelled primers leads to the generation of distinct restrictionfragments, which can be used to identify their associated mRNA. Suchmethods include that described in U.S. Pat. No. 5,712,126 whichdiscloses a method of introducing adapters into restriction endonucleasedigested cDNA fragments which allow selective amplification andlabelling of 3′ terminal cDNA fragments. Similarly, WO 99/02727,discloses a method of amplifying 3′ terminal restriction fragments usingsolid phase supports and PCR primers which probe the unknown sequenceadjacent to a known restriction site. In this technology cDNAs areprepared using biotinylated anchor primers which ensures that all cDNAsare terminated with a short poly-A tail of fixed length and can beimmobilised on a solid phase substrate. The poly-T primer mayadditionally carry a primer sequence at its 5′ terminus. The capturedcDNAs are then cleaved with an ordinary type II restrictionendonuclease. An adapter is ligated to the resulting known sticky-end.The adapter is designed to carry a primer sequence. The resulting doublestranded construct is then denatured. The strand that is not immobilisedcan be washed away if desired. A family of primers complementary to theadapter primer with an overlap of 4 bases into the unknown sequenceadjacent to the adapter primer is added to the denatured mixture. With a4 base overlap there are 256 possible primers. To identify the probes,they are tagged with mass labels using a cleavable linker, so that eachof the 256 possible 4 bp overlaps is identified by a label that isuniquely identifiable in a mass spectrometer. This results in apopulation of fragments with varying lengths according to where theordinary type II restriction endonuclease cut them and with one of 256possible mass labelled primers at the 5′ terminus of the cDNA. The cycleof denaturing and primer extension can be performed as many times asdesired. If only the adapter primer sites are used, a linearamplification can be performed. This causes smaller distortion of cDNAquantification than exponential amplification. If exponentialamplification is desired then the poly-T oligos used to trap the mRNAsmust carry a primer site as well. Exponential amplification may bedesirable if small tissue samples must be analysed despite the potentialfor distortions of cDNA frequencies.

Again, the mass labelled 3′ restriction fragments are separated on thebasis of the length, using capillary electrophoresis, of the restrictionfragments followed by analysis of the mass labels at the termini of thecDNA fragments. This technique, like that disclosed in WO 98/48047 ispreferably practised with 256 mass labels and would thus benefit in thesame way from the advantageous features of the mass labels of thisinvention.

Thus, in a further aspect of this invention, there is provided a methodof analysis comprising the steps of:

-   -   1. Providing a population of mass labelled nucleic acid        fragments of different lengths, where the mass labels are        indicative of a feature of the labelled nucleic acids.    -   2. Separating the labelled fragments on the basis of their size    -   3. Detaching the mass labels from the labelled fragments    -   4. Detecting the mass labels in a mass spectrometer.

In certain embodiments of this aspect of the invention, the assaydetermines the sequence of nucleic acid or a series of nucleic acids. Insequencing embodiments based on the generation of Sanger ladders themass label identifies the terminating nucleotide of each fragment andeach fragment is identified by a set of four labels. In Sangersequencing embodiments the labels are introduced as mass labelledprimers or labelled terminating nucleotides.

In other embodiments of this aspect of the invention, the assay is usedto determine the identity and quantity of expressed RNA molecules. Inpreferred embodiments, the mass labelled nucleic acids are generatedaccording to the methods disclosed in WO 98/48047 or WO 99/02727. Inembodiments using these methods mass labels are introduced into thenucleic acid fragments by ligation of mass labelled adapters or byextension of mass labelled primers, respectively. For one of ordinaryskill in the art, it should be clear that other methods of geneexpression profiling based on the size of nucleic acid fragments, suchas those disclosed in PCT/GB93/0145, EP-A-0 735 144 or U.S. Pat. No.5,712,126, can be adapted for use with the labels of this invention.

In preferred embodiments of this invention the step of separating theanalytes on the basis of size is carried out using capillaryelectrophoresis or high performance liquid chromatography, using, forexample, systems such as those provided by Transgenomic, Inc. (San Jose,Calif., USA.) and disclosed in U.S. Pat. No. 5,585,236, U.S. Pat. No.5,772,889 and other applications. Preferably the separation is performedon-line with a mass spectrometer.

In preferred embodiments, the step of detaching the mass labels fromtheir associated analytes takes place within the ion source of the massspectrometer. Linkers that allow a mass label to be easily cleaved fromits associated analyte in a mass spectrometer ion source are disclosedin PCT/GB98/00127. Compounds that improve the sensitivity of detectionof a mass label by mass spectrometry are disclosed in PCT/GB98/00127.

For one of ordinary skill in the art, it should be clear that othersizing assays could be adapted for use with the mass labels of thisinvention, including, for example, the multiplexed genotyping assaydisclosed by Grossman P. D. et al. in Nucleic Acids Research 1994 Oct.25;22(21):4527-34. This assay would benefit greatly from the ability tomultiplex to higher orders and still resolve the size of fragmentseasily.

Protein Expression Profiling and 2-Dimensional Gel Electrophoresis

Techniques for profiling proteins, that is to say cataloguing theidentities and quantities of all the proteins expressed in a tissue, arenot well developed in terms of automation or throughput. The classicalmethod of profiling a population of proteins is by two-dimensionalelectrophoresis (R. A. Van Bogelen., E. R. Olson, “Application oftwo-dimensional protein gels in biotechnology”, Biotechnol. Ann. Rev.,1:69-103, 1995). In this method a protein sample extracted from abiological sample is separated on a narrow gel strip. This firstseparation usually separates proteins on the basis of their iso-electricpoint. The entire gel strip is then laid against one edge of arectangular gel, such as a polyacrylamide gel. The separated proteins inthe strip are then electrophoretically separated in the second gel onthe basis of their size, e.g. by Sodium Dodecyl Sulphate PolacrylamideGel Electrophoresis (SDS PAGE). This methodology is slow and verydifficult to automate. It is also relatively insensitive in its simplestincarnations. Once the separation is complete the proteins must bevisualised. This typically involves staining the gel with a reagent thatcan be detected visually or by fluorescence. Radiolabelling andautoradiography are also used. In other methods fluorescent dyes may becovalently linked to proteins in a sample prior to separation. Covalentaddition of a dye can alter the mobility of a protein and so this issometimes less preferred, particularly if comparisons are to be madewith public databases of 2-dimensional gel images. Having visualised theproteins in a gel it is usually necessary to identify the proteins inparticular spots on the gel. This is typically done by cutting the spotsout of the gel and extracting the proteins from the gel matrix. Theextracted proteins can then be identified by a variety of techniques.Preferred techniques involve digestion of the protein, followed bymicrosequencing. A number of improvements have been made to increaseresolution of proteins by 2-D gel electrophoresis and to improve thesensitivity of the system. One method to improve the sensitivity of 2-Dgel electrophoresis and its resolution is to analyse the protein inspecific spots on the gel by mass spectrometry (Jungblut P., Thiede B.“Protein identification from 2-D gels by MALDI mass spectrometry”, MassSpectrom. Rev. 16, 145-162, 1997). One such method is in-gel trypticdigestion followed by analysis of the tryptic fragments by massspectrometry to generate a peptide mass fingerprint. If sequenceinformation is required, tandem mass spectrometry analysis can beperformed.

At present 2-D analysis is a relatively slow “batch” process. It is alsonot very reproducible and it is expensive to analyse a gel. Since mostof the costs in a gel based analysis are in the handling of each gel itwould be desirable to be able to multiplex a number of samples on a 2-Dgel simultaneously. If it were possible to label the proteins indifferent samples with a different, independently detectable tag, thenthe proteins in each sample could be analysed simultaneously on the samegel. This would be especially valuable for studies where it is desirableto follow the behaviour of the same proteins in a particular organism atmultiple time points, for example in monitoring how a bacteria respondsto a drug over a predetermined time course. Similarly comparing biopsymaterial from multiple patients with the same disease with correspondingcontrols would be desirable to ensure that the same protein fromdifferent samples would end up at the same spot on the gel. Running allthe samples on the same gel would allow different samples to be comparedwithout having to be concerned about the reproducibility of theseparation of the gel. To achieve this requires a series of labels whoseeffect on the mobility of the proteins in different samples will be thesame, so that a particular protein which is labelled with a differentlabel in each sample will still end up at the same position in the gelirrespective of its label.

More recently attempts have been made to exploit mass spectrometry toanalyse whole proteins that have been fractionated by liquidchromatography or capillary electrophoresis (Dolnik V. “Capillary zoneelectrophoresis of proteins”, Electrophoresis 18, 2353-2361, 1997).In-line systems exploiting capillary electrophoresis mass spectrometryhave been tested. The analysis of whole proteins by mass spectrometry,however, suffers from a number of difficulties. The first difficulty isthe analysis of the complex mass spectra resulting from multipleionisation states accessible by individual proteins. The second majordisadvantage is that the mass resolution of mass spectrometers is atpresent quite poor for high molecular weight species, i.e. for ions thatare greater than about 4 kilodaltons in mass so resolving proteins thatare close in mass is difficult. A third disadvantage is that furtheranalysis of whole proteins by tandem mass spectrometry is difficult asthe fragmentation patterns for whole proteins are extremely complex anddifficult to interpret.

PCT/GB98/00201 and PCT/GB99/03258, describe methods of characterisingcomplex mixtures of proteins by isolating C-terminal peptides from theproteins in the mixtures and analysing them by mass spectrometry. Themethods described can be used to determine whether proteins are presentor absent in a sample but would not give comparative data betweensamples. The methods do not describe techniques for analysis of multiplesamples simultaneously, which would be necessary for quantitativecomparison of protein expression levels in multiple samples.

EP-A-0 594 164 describes a method of isolating a C-terminal peptide froma protein in a method to allow sequencing of the C-terminal peptideusing N-terminal sequencing reagents. In this method the protein ofinterest is digested with an endopeptidase which cleaves at theC-terminal side of lysine residues. The resultant peptides are reactedwith DITC polystyrene, which reacts with all free amino groups.N-terminal amino groups that have reacted with the DITC polystyrene canbe cleaved with trifluoroacetic acid (TFA) thus releasing the N-terminusof all peptides. The epsilon-amino group of lysine is not cleaved,however, and all non-terminal peptides are thus retained on the supportand only C-terminal peptides are released. According to this document,the C-terminal peptides are recovered for micro-sequencing.

Nature Biotechnology 17: 994-999 (1999) discloses the use of “isotopeencoded affinity tags” for the capture of peptides from proteins toallow protein expression analysis. In this article, the authors describethe use of a biotin linker, which is reactive to thiols to capturepeptides with cysteine in them. A sample of protein from one source isreacted with the biotin linker and cleaved with an endopeptidase. Thebiotinylated cysteine containing peptides can then be isolated onavidinated beads for subsequent analysis by mass spectrometry. Twosamples can be compared quantitatively by labelling one sample with thebiotin linker and labelling the second sample with a deuterated form ofthe biotin linker. Each peptide in the samples is then represented as apair of peaks in the mass spectrum where the relative peak heightsindicate their relative expression levels.

The method in this paper has a number of limitations. Of the variouslimitations to this “isotope encoding” method, the first is the relianceon the presence of thiols in a protein—many proteins do not have thiolswhile others have several. In a variation on this method, linkers may bedesigned to react with other side chains such as amines, but since manyproteins contain more than one lysine residue, multiple peptides perprotein will be isolated in this approach. It is likely that this wouldnot reduce the complexity of the sample sufficiently for analysis bymass spectrometry. A sample that contains too many species is likely tosuffer from “ion suppression” in which certain species ionisepreferentially over other species which would normally appear in themass spectrum in a less complex sample. In general, capturing proteinsby their side chains may give either too many peptides per protein orcertain proteins will be missed altogether.

The second limitation of this approach is in the method used to comparethe expression levels of proteins from different samples. Labelling eachsample with a different isotope variant of the affinity tag results inan additional peak in the mass spectrum for each peptide in each sample,which means that if two samples are analysed together there will betwice as many peaks in the spectrum. Similarly, if three samples areanalysed together, the spectrum will be three times more complex thanfor one sample alone. It might be feasible to attempt the comparison oftwo or three samples by this approach but this may well be the limit asthe ever increasing numbers of peaks will increase the likelihood thattwo different peptides will have overlapping peaks in the mass spectrum.

A further limitation reported by the authors of the above paper is themobility change caused by the tags. The authors report that peptideslabelled with the deuterated biotin tag elute slightly after the samepeptide labelled with the undeuterated tag.

In view of the above, a further aim of the present invention it is an toprovide an improved method of determining the identity and relativequantities of polypeptides in a number of samples of complex polypeptidemixtures simultaneously. It is a further aim of this aspect of theinvention to ensure that all proteins are represented in the analysis.It is also an aim of this aspect of the invention to provide mass labelsand techniques which allow multiple samples to be analysedsimultaneously and quantitatively without increasing significantly thecomplexity of the mass spectrum when compared to the spectrum that wouldbe obtained from a single sample alone. It is a final aim of this aspectof the invention to provide labels which have the same effect on themobility of the labelled peptide, so that samples of the same peptidelabelled with different tags will co-elute after a chromatographicseparation.

Thus, a further preferred embodiment of this invention provides a methodof analysing a protein sample containing more than one protein, themethod comprising the steps of:

-   -   1. Labelling peptides, polypeptides and/or proteins in the        sample with at least one discretely resolvable mass label from        the sets and arrays of this invention, such that each peptide,        polypeptide and/or protein is labelled with a label or        combination of labels unique to that protein    -   2. Analysing the labelled peptides, polypeptides and/or proteins        by mass spectrometry, preferably according to an aspect of this        invention e.g. tandem mass spectrometry, to detect the labels        attached to the proteins. The labelled peptides in the sample        may then be identified and their relative expression levels        determined.

It is preferred that multiple samples are subjected to the aboveprocess. It is further preferred that for each of a number of samples,prior to labelling step (1) above, peptides are isolated frompolypeptides in the mixture using a cleavage agent, especially asequence specific cleavage agent. After labelling step (1), the samplesmay be pooled, if desired. Optionally, after labelling step (1) and/orpooling the samples, the peptides polypeptides and/or proteins in thesample or samples may be separated, by gel electrophoresis, iso-electricfocusing, liquid chromatography or other appropriate means, preferablygenerating discrete fractions. These fractions may be bands or spots ona gel or liquid fractions from a chromatographic separation. Fractionsfrom one separation may separated further using a second separationtechnique. Similarly further fractions may be fractionated again untilthe proteins are sufficiently resolved for the subsequent analysissteps.

This aspect of the invention thus provides a further application of thelabels and methods of this invention described above. A set or array oflabels of the present invention can be used to increase the throughputof a 2-D gel electrophoresis analysis of the proteins in an organism.Each of the mass labels alters the mobility of its associated protein inthe same way but is still independently detectable. In known uses ofmass spectrometry to analyse proteins from a 2-D gel, such as peptidemass fingerprinting, it is required that the proteins be extracted fromthe gel and be purified to remove detergents such as SDS and othercontaminants from the gel. The labels of this invention allow relativelyunpurified extract of proteins from the gel to be introduced directlyinto the mass spectrometer and the associated labels can then beidentified by the methods of this invention in a background ofcontaminating material.

In a particularly preferred embodiment of this aspect of the inventionmultiple samples are subjected to the following process:

-   -   1. for each of a number of samples, isolating peptides from        polypeptides in the mixture using sequence specific cleavage        reagents;    -   2. labelling the isolated peptides in each sample with the        labels of this invention such that each sample is identified by        a unique label;    -   3. pooling the labelled samples;    -   4. optionally separating the pooled and labelled peptides        chromatographically or electrophoretically;    -   5. analysing these labelled samples by tandem mass spectrometry        to identify the labelled peptides in the sample and determine        their relative expression levels.

Another preferred embodiment of this aspect of the invention provides amethod of analysing a series of protein samples each sample containingmore than one protein, the method comprising the steps of:

-   -   1. Covalently reacting the proteins of each of the samples with        at least one discretely resolvable mass label from the sets and        arrays of this invention, such that the proteins of each sample        are labelled with one or more mass labels that are different        from the labels reacted with the proteins of every other sample.    -   2. Pooling the mass labelled samples.    -   3. Separating the pooled samples by gel electrophoresis,        iso-electric focusing, liquid chromatography or other        appropriate means to generate discrete fractions. These        fractions may be bands or spots on a gel or liquid fractions        from a chromatographic separation. Fractions from one separation        may separated further using a second separation technique.        Similarly further fractions may be fractionated again until the        proteins are sufficiently resolved for the subsequent analysis        steps.    -   4. Analysing the fractions by mass spectrometry, preferably        according to an aspect of this invention, to detect the labels        attached to the proteins.

A still further preferred embodiment of this aspect of the presentinvention provides a method of identifying a protein in a samplecontaining more than one protein, the method comprising the steps of:

-   -   1. Covalently reacting the proteins of the sample with at least        one discretely resolvable mass label from the sets and arrays of        this invention.    -   2. Separating the proteins by gel electrophoresis, iso-electric        focusing, liquid chromatography or other appropriate means to        generate discrete fractions. These fractions may be bands or        spots on a gel or liquid fractions from a chromatographic        separation. Fractions from one separation may separated further        using a second separation technique. Similarly further fractions        may be fractionated again until the proteins are sufficiently        resolved for the subsequent analysis steps.    -   3. Digesting the proteins in the fraction with a sequence        specific cleavage reagent.    -   4. Optionally reacting the proteins in the sample with an        additional mass label    -   5. Analysing the digested fractions by liquid chromatography        mass spectrometry where the elution time of mass marked peptides        from the liquid chromatography column step is determined by        detecting the mass labels attached to the peptides. A mass        spectrometry analysis is performed, preferably according to an        aspect of this invention, to detect the labels attached to the        proteins.    -   6. Comparing the elution profile of the labelled peptides from        the liquid chromatography mass spectrometry analysis of step 5        with profiles in a database to determine whether the protein has        been previously identified.

A yet further preferred embodiment of this aspect of the presentinvention provides a method of identifying a protein from a series ofprotein samples each sample containing more than one protein, the methodcomprising the steps of:

-   -   1. Covalently reacting the proteins of each of the samples with        at least one discretely resolvable mass label from the sets and        arrays of this invention, such that the proteins of each sample        are labelled with one or more mass labels that are different        from the labels reacted with the proteins of every other sample.    -   2. Pooling the mass labelled samples.    -   3. Separating the proteins by gel electrophoresis, iso-electric        focusing, liquid chromatography or other appropriate means to        generate discrete fractions. These fractions may be bands or        spots on a gel or liquid fractions from a chromatographic        separation. Fractions from one separation may separated further        using a second separation technique. Similarly further fractions        may be fractionated again until the proteins are sufficiently        resolved for the subsequent analysis steps.    -   4. Digesting the proteins in the fraction with a sequence        specific cleavage reagent to generate characteristic peptides        for each protein in the sample.    -   5. Optionally reacting the proteins in the sample with an        additional mass label.    -   6. Analysing the digested fractions by liquid chromatography        mass spectrometry where the elution time of mass marked peptides        from the liquid chromatography column step is determined by        detecting the mass labels attached to the peptides. A mass        spectrometry analysis is performed, preferably according to an        aspect of this invention, to detect the labels attached to the        proteins.    -   7. Comparing the elution profile of the labelled peptides from        the liquid chromatography mass spectrometry analysis of step 6        with profiles in a database to determine whether the protein has        been previously identified.

Step 1 of the above preferred embodiments of this invention involvescovalently reacting a mass label of this invention to the reactive sidechains of a population of proteins. It is well known in the art that thereactive side-chain functionalities can be selectively reacted. Reactiveside-chains include lysine, serine, threonine, tyrosine and cysteine.Cysteine is often cross-linked with itself to form disulphide bridges.For the purposes of this invention it is not essential that thesebridges be broken but cysteine side chains can be highly reactive andmay be readily reacted with a variety of reagents. If disulphide bridgesare present, these can be broken by reducing the disulphide bridge to apair of thiols with mercaptethanol. Thiols can be selectively capped byiodoacetate (Aldrich) under mildly basic conditions which promote theformation of a thiolate ion (Mol. Microbiol. 5: 2293, 1991). Anappropriate mild base is a carbonate. For the proposes of thisinvention, a mass label of this invention whose reactive functionalityis an iodoacetyl group can be reacted with the thiols of an analyteprotein. In other embodiments the population of proteins may be treatedwith a mass marker whose reactive functionality is an isocyanate group.Isocyanates will react almost exclusively with the alpha-amino group atthe N-terminus of the proteins and with any lysine epsilon-amino groups,i.e. with primary amines under mild conditions, i.e. at room temperaturein a neutral solvent to give a urea derivative. These reagents can alsobe made to react with any hydroxyl bearing side-chains, such as serine,threonine and tyrosine side chains, at higher temperatures in thepresence of an appropriate catalyst such as pyridine or a tin compoundsuch as dibutyl stannyl laurate to give a urethane derivative. In analternative embodiment the population of proteins can be treated with amass marker whose reactive functionality is a silyl group such aschlorosilane. These compounds react readily with most reactivefunctional groups. Amine derivatives are not stable under aqueousconditions and so can be hydrolysed back to the free amine if that isdesired. Sulphonyl chlorides can also be used as a reactive group on amass label to selectively react the mass label with free amines such aslysine. Carboxylic acid side chains could also be reacted with thelabels of this invention although it is usually necessary to activatethese side chains to ensure that they will react. Acetic anhydride iscommonly used for this purpose. This forms mixed anhydrides at freecarboxylic acids which can then be reacted with a nucleophilicfunctionality such as amine.

The above specific embodiments are intended only as examplesillustrating preferred methods of selectively reacting side-chainfunctionalities with mass labels. A wide variety of reactive groups areknown in the art and many of these can be used to complete the firststeps of these aspects of this invention. It may also be desirable toreact more than one type of side chain of the proteins in a sample withdifferent mass labels. If multiple samples are to be analysedsimultaneously then two or more labels can be used to label each sample.This allows more information to be derived from each protein to aid inits identification.

In step 3 and step 4 of the latter two embodiments of this aspect of theinvention, the C-terminally modified proteins are then treated with asequence specific cleavage agent. In some embodiments sequence specificendoproteinases such as trypsin, chymotrypsin, thrombin or other enzymesmay be used. Cleavage agents may alternatively be chemical reagents.These are preferably volatile to permit easy removal of unreactedreagent. Appropriate chemical cleavage reagents include cyanogen bromidewhich cleaves at methionine residues and BNPS-skatole which cleaves attryptophan residues (D. L. Crimmins et al., Anal. Biochem. 187: 27-38,1990).

In the above preferred embodiments of this aspect of the invention, thestep of fractionating the proteins is preferably effected by performing2-dimensional gel electrophoresis, using iso-electric focusing in thefirst dimension and SDS PAGE in the second dimension. The gel is thenvisualised to identify where proteins have migrated to on the gel. Thespots can then be excised from the gel and the proteins are thenextracted from the excised gel spot. These extracted proteins may thenbe analysed directly by electrospray mass spectrometry or some othersuitable ionisation procedure. Alternatively further fractionation maybe performed in-line with the mass spectrometer such as HPLC massspectrometry.

In step 3 and step 4 of the latter two preferred embodiments of thisaspect of the invention, the digested proteins are optionally reactedwith an additional mass label of this invention. This is of moresignificance to the latter preferred embodiment of this invention, wheremultiple samples are analysed simultaneously. Most enzymatic digestionsand some of the chemical cleavage methods leave free amines on theresultant peptides of the digested fractionated proteins which can bereacted with a mass label. This means that the same label will appear onall peptides and can be detected selectively to maximise the sensitivityof this analysis.

In step 6 and step 7 of the latter two preferred embodiments of thisaspect of the invention, the elution profile of the peptides generatedby digesting the fractionated proteins is used to search a pre-formeddatabase to determine whether the proteins have been previouslyidentified. The peptides eluting from the liquid chromatography columninto a mass spectrometer may be further analysed by tandem massspectrometry to determine sequence information which can be used toidentify proteins. Peptide sequence data can be used to search a proteinsequence database or can translated into nucleic acid sequence data tosearch nucleic acid sequence databases.

Isolation of Post-translationally Modified Peptides

Carbohydrates are often present as a post-translational modification ofproteins. These carbohydrates often have carbonyl groups. Carbonylgroups can be tagged allowing proteins bearing such modifications to bedetected or isolated. Biocytin hydrazide (Pierce & Warriner Ltd,Chester, UK) will react with carbonyl groups in a number of carbohydratespecies (E. A. Bayer et al., Anal. Biochem. 170, 271-281, “Biocytinhydrazide—a selective label for sialic acids, galactose, and othersugars in glycoconjugates using avidin biotin technology”, 1988).Proteins bearing carbohydrate modifications in a complex mixture canthus be biotinylated. The protein mixture may then be treated with anendoprotease, such as trypsin, to generate peptides from the proteins.Biotinylated, hence carbohydrate modified, peptides may then be isolatedusing an avidinated solid support. A series of samples may be treated inthis way and the peptides obtained may be reacted with the mass labelsof this invention, such that peptides from each sample bear a mass labelor combination of mass labels relatable to the peptide or peptides fromthat sample. Preferably peptides from each sample bear a different masslabel. These mass tagged carbohydrate-bearing peptides may then beanalysed by liquid chromatography tandem mass spectrometry.

A number of research groups have reported on the production ofantibodies, which bind to phosphotyrosine residues in a wide variety ofproteins (see for example A. R. Frackelton et al., Methods Enzymol. 201,79-92, “Generation of monoclonal antibodies against phosphotyrosine andtheir use for affinity purification of phosphotyrosine-containingproteins”, 1991 and other articles in this issue of Methods Enzymol.).This means that a significant proportion of proteins that have beenpost-translationally modified by tyrosine phosphorylation may beisolated by affinity chromatography using these antibodies as theaffinity column ligand.

These phosphotyrosine binding antibodies can be used in the context ofthis invention to isolate peptides containing phosphotyrosine residues.Thus proteins in a complex mixture may be treated with a sequencespecific endopeptidase to generate free peptides. These may then bepassed through an anti-phosphotyrosine antibody column, which willretain peptides containing a phosphotyrosine group. A series of samplesmay be treated in this way and the peptides obtained may be reacted withthe mass tags of this invention, such that peptides from each samplebear a mass label or combination of mass labels relatable to the peptideor peptides from that sample. Preferably peptides from each sample beara different mass label. These mass labelled phosphotyrosine-bearingpeptides may then be analysed by liquid chromatography tandem massspectrometry.

Isolation of Terminal Peptides from Proteins

A preferred method of protein expression profiling according to thepresent invention is to isolate only one peptide from each protein inthe sample. Provided that the isolated peptide fragment is of sufficientlength, the fragment will be specific to its parent protein. In thefirst step of this aspect of the present invention, peptides areisolated from each protein in each of a number of samples of complexprotein mixtures. In some embodiments of this aspect it is preferredthat terminal peptides are isolated. Isolation of terminal peptidesensures that at least one and only one peptide per protein is isolated.Methods for isolating peptides from the termini of polypeptides arediscussed in PCT/GB98/00201 and PCT/GB99/03258.

Thus, this aspect of the present invention provides a method of proteinprofiling, which method comprises:

-   -   (a) treating a sample comprising a population of a plurality of        polypeptides with a cleavage agent which is known to recognise        in polypeptide chains a specific amino acid residue or sequence        and to cleave at a cleavage site, whereby the population is        cleaved to generate peptide fragments;    -   (b) isolating a population of peptide fragments bearing as a        reference terminus the N-terminus or the C-terminus of the        polypeptide from which they were fragmented, each peptide        fragment bearing at the other end the cleavage site proximal to        the reference terminus;    -   (c) prior to or after isolating the peptide fragments, labelling        each reference terminus of the polypeptides with a mass label,        or a combination of mass labels from a set or an array of mass        labels of the present invention, wherein each reference terminus        is relatable to its label or combination of labels; and    -   (d) determining by mass spectrometry a signature sequence of one        or more of the isolated fragments, which signature sequence is        the sequence of a pre-determined number of amino acid residues        running from the cleavage site;        wherein a signature sequence characterises each polypeptide.

An alternative preferred method provided by this aspect of the presentinvention makes use of a second cleavage agent to generate furtherfragments, which may themselves be identified and used to characterisetheir parent polypeptide or protein. This method comprises:

-   (a) contacting a sample comprising one or more polypeptides with a    first cleavage agent to generate polypeptide fragments;-   (b) isolating one or more polypeptide fragments, each fragment    comprising the N-terminus or the C-terminus of the polypeptide from    which it was fragmented;-   (c) prior to or after isolating the polypeptide fragments, labelling    each terminus of the polypeptides with a mass label, or a    combination of mass labels from a set or an array of mass labels of    the present invention, wherein each terminus is relatable to its    label or combination of labels; and-   (d) identifying the isolated fragments by mass spectrometry;-   (e) repeating steps (a)-(d) on the sample using a second cleavage    agent that cleaves at a different site from the first cleavage    agent; and-   (f) characterising the one or more polypeptides in the sample from    the fragments identified in steps (d) and (e).

In both of the above methods, the step of labelling the referencetermini can take place before or after isolating the fragments and canalso take place before the fragments are cleaved from their parentpolypeptides or proteins, if desired.

Regarding the isolation of peptide fragments, in preferred embodimentsof this aspect of the present invention, terminal peptides may beisolated from a complex mixture of proteins using a method, comprisingthe steps of:

-   -   1. Digesting the complex mixture of proteins completely with a        Lys-C specific cleavage enzyme, i.e. a reagent that cuts at the        peptide bond immediately adjacent to a lysine residue on the        C-terminal side of that residue.    -   2. Contacting the resultant peptides with an activated solid        support that will react with free amino groups.    -   3. Optionally reacting the captured peptides with a bifunctional        reagent, which has at least one amine reactive functionality.    -   4. Contacting the captured peptides with a reagent that which        cleaves at the alpha amino groups of each peptide on the        support. All peptides that are not C-terminal will have a lysine        residue covalently linking them to the solid support. Thus free        C-terminal peptides are selectively released.    -   5. Optionally contacting the released peptides with a second        solid support that will react with the second reactive        functionality of the bifunctional reagent used in step 3 to        capture any peptides that did not react properly with the first        support.    -   6. Recovering the peptides remaining free in solution.

In preferred embodiments of this method, the proteins in the complexmixture are denatured, reduced and treated with a reagent to cap thiolsin the proteins. Typical protocols involve denaturing the proteins in abuffer at pH 8.5 with a high concentration of guanidine hydrochloride(6-8 M), as a denaturation reagent, in the presence of an excess ofmercaptoethanol or dithiothreitol, as reducing agents, and an excess ofa capping agent such as vinylpyridine.

In step 1 of this method, the complex mixture of proteins is completelydigested with a Lys-C specific cleavage enzyme, which may be, forexample, endopeptidase Lys-C from Lysobacter enzyrnogenes (BoehringerMannheim).

In step 2 of this method, the resultant peptides are contacted with asolid support that reacts with amines. In preferred embodiments thesolid phase support is derivitised with an isothiocyanate compound. Inone embodiment the peptide population is reacted with isothiocyanatoglass (DITC glass, Sigma-Aldrich Ltd, Dorset, England) in the presenceof a base. This captures all peptides to the support through any freeamino groups.

Step 3 is optional but preferred. It may be difficult to guarantee thatall non-C-terminal peptides will react completely with the first solidsupport at both the lysine side-chain amino-group and the N-terminalalpha amino-group. Peptides that react only through the lysineside-chain amino groups will remain attached to the support insubsequent steps. Peptides that react only through their alpha-aminogroup will be cleaved from the support along with C-terminal peptide.This step allows the non-C-terminal peptides to be distinguished fromC-terminal peptides. In this optional step, the bifunctional reagent maybe N-succinimidyl[4-vinylsulphonyl]benzoate (SVSB from Pierce & WarrinerLtd., Chester, UK). This compound comprises an amine-reactiveN-hydroxysuccinimide ester linked to a thiol-reactive vinyl sulphonemoiety. The compound reacts very easily with amines via the esterfunctionality without reaction of the vinyl sulphone and can beseparately reacted with thiols at a later stage. Thus the SVSB isreacted with any free amines on the support under slightly basicconditions. In the presence of a large excess of the SVSB compound andgiven that the peptides on the support are immobilised, the SVSB willreact with the peptides only through the succinimide functionalityleaving the vinyl sulphone moiety free for further reaction. Inalternative embodiments, any unreacted amines may be reacted with biotincoupled to an amine reactive functionality such as N-hydroxysuccinimide(NHS) biotin (Sigma-Aldrich Ltd, Dorset, England). This allowsincompletely reacted peptides to be captured later on avidinated beadsor on an avidinated resin in an affinity capture column.

In step 4 of this method the captured peptides are contacted with areagent that cleaves at the alpha amino groups of each peptide on thesupport. In embodiments where DITC glass is used as the amine reactivesupport, the peptides are treated with an appropriate volatile acid suchas trifluoroacetic acid (TFA) which cleaves the N-terminal amino acidfrom each peptide on the support. All peptides that are not C-terminalwill have a lysine residue covalently linking them to the solid support.Thus free C-terminal peptides are selectively released.

The optional step 5 is preferred especially if the optional step 3 isperformed. The non-C-terminal peptides that do not react completely withthe amine reactive support are removed by this step. If SVSB is used totag non-C-terminal peptides that only reacted through their alpha-aminogroups they will have a reactive functionality available which willallow them to be reacted with a solid support derivitised with anappropriate nucleophile, preferably a thiol. If DITC glass is used instep 4, which is preferred, then the peptides may be released fromsupport using TFA. The released peptides may be recovered astrifluoroacetate salts by evaporating the TFA away. The peptides maythen be resuspended in a buffer with a pH of about 7 or just in anappropriate neutral solvent such as dimethylformamide,dimethylsulphoxide or a mixture of water and acetone. The peptides arethen added to the thiol derivitised support. At pH 7 the remaining vinylfunctionality on the SVSB treated peptides should react almostexclusively with the thiol support rather than with free amines exposedby cleavage of the peptides from the DITC glass support. Thiolderivitised Tentagels are available from Rapp Polymere GmbH (Tübingen,Germany) or a thiol derivitised support can be prepared by incubating asilica gel with 3-mercaptopropyltrimethoxysilane.

In step 6 of this method the released peptides are recovered. Ifoptional steps 3 and 5 are used the peptides may be present in a varietyof solvents or buffers. These will be selected to be volatile inpreferred embodiments. If the peptides are recovered directly from thefirst support, which is DITC glass in preferred embodiments, then it islikely that the peptides will be in TFA, which is volatile. The peptidesare preferably recovered from these volatile solvents or buffers byevaporating the solvent or buffer. The peptides isolated by this methodwill have a free alpha-amino group available for reaction with thelabels of this invention.

Labelling Isolated Peptides

Any of the mass labels of the present invention can be used in theprotein expression profiling embodiments described in this aspect of theinvention. The mass labels illustrated in FIGS. 22 and 23 areparticularly preferred for use with this invention, especially thisaspect of the present invention. These compounds have a vinyl sulphonereactive group, which will allow these compounds to undergo additionreactions with free amines and thiols. If only one label is desired perpeptide then the proteins in the complex mixtures may be treated withcapping agents prior to cleavage with the sequence specificendopeptidase. Phenyl, ethyl and methyl vinyl sulphone will react withfree amines and thiols capping them while still permitting cleavage bytrypsin of the capped proteins. The epsilon amine residues of lysinewill react with two vinyl sulphone moieties if the vinyl sulphonemoieties are not hindered, particularly ethyl and methyl vinyl sulphone.

After attachment of the markers these labelled peptides will have a massthat is shifted by the mass of the label. The mass of the peptide may besufficient to identify the source protein. In this case only the labelneeds to be detected which can be achieved by selected reactionmonitoring with a triple quadrupole, discussed in more detail below.Briefly, the first quadrupole of the triple quadrupole is set to letthrough ions whose mass-to-charge ratio corresponds to that of thepeptide of interest, adjusted for the mass of the marker. The selectedions are then subjected to collision induced dissociation (CID) in thesecond quadrupole. Under the sort of conditions used in the analysis ofpeptides the ions will fragment mostly at the amide bonds in themolecule. The markers in FIGS. 22 and 23 have an amide bond, whichreleases the terminal pre-ionised portion of the tag on cleavage.Although the tags all have the same mass, the terminal portion isdifferent because of differences in the substituents on either side ofthe amide bond. Thus the markers can be distinguished from each other.The presence of the marker fragment associated with an ion of a specificmass should confirm that the ion was a peptide and the relative peakheights of the tags from different samples will give information aboutthe relative quantities of the peptides in their samples. If the mass isnot sufficient to identify a peptide, either because a number ofterminal peptides in the sample have the same terminal mass or becausethe peptide is not known, then sequence information maybe determined byanalysis of the complete CID spectrum. FIG. 24 shows a theoreticalspectrum for two samples of a peptide with the sequenceH₂N-gly-leu-ala-ser-glu-COOH (SEQ ID NO: 1), where each sample isattached to one of the labels with the formulae shown in FIG. 23. Thespectrum is idealised, as it only shows the b-series fragments and doesnot show other fragmentations or any noise peaks, however it doesillustrate that the spectrum is clearly divided into a higher massregion corresponding to peptide fragmentation peaks and a lower massregion corresponding to mass label peaks. If desired, the peptidefragmentation peaks can be used to identify the peptides while the masstag peaks give information about the relative quantities of thepeptides.

Separation of Labelled Peptides by Chromatography or Electrophoresis

Preferably in this aspect of the invention, in the step prior to massspectroscopic analysis the labelled terminal peptides are subjected to achromatographic separation prior to analysis by mass spectrometry. Thisis preferably High Performance Liquid Chromatography (HPLC) which can becoupled directly to a mass spectrometer for in-line analysis of thepeptides as they elute from the chromatographic column. A variety ofseparation techniques may be performed by HPLC but reverse phasechromatography is a popular method for the separation of peptides priorto mass spectrometry. Capillary zone electrophoresis is anotherseparation method that may be coupled directly to a mass spectrometerfor automatic analysis of eluting samples. These and other fractionationtechniques may be applied to reduce the complexity of a mixture ofpeptides prior to analysis by mass spectrometry.

Protein Quantification and Identification by Tandem Mass Spectrometry

In the method of this aspect of the invention, the labelled isolatedpeptides are analysed by tandem mass spectrometry.

As discussed earlier tandem mass spectrometers allow ions with apre-determined mass-to-charge ratio to be selected and fragmented, e.g.by collision induced dissociation (CID). The fragments can then bedetected providing structural information about the selected ion. Whenpeptides are analysed by CID in a tandem mass spectrometer,characteristic cleavage patterns are observed, which allow the sequenceof the peptide to be determined. Natural peptides typically fragmentrandomly at the amide bonds of the peptide backbone to give series ofions that are characteristic of the peptide. CID fragment series areusually denoted a_(n), b_(n), c_(n), etc. for cleavage at the n^(th)peptide bond, where the charge of the ion is retained on the N-terminalfragment of the ion. Similarly, fragment series are denoted x_(n),y_(n), z_(n), etc. where the charge is retained on the C-terminalfragment of the ion. This notation is depicted in the following Scheme1:

Trypsin and thrombin are favoured cleavage agents for tandem massspectrometry as they produce peptides with basic groups at both ends ofthe molecule, i.e. the alpha-amino group at the N-terminus and lysine orarginine side-chains at the C-terminus. This favours the formation ofdoubly charged ions, in which the charged centres are at oppositetermini of the molecule. These doubly charged ions produce bothC-terminal and N-terminal ion series after CID. This assists indetermining the sequence of the peptide. Generally speaking only one ortwo of the possible ion series are observed in the CID spectra of agiven peptide. In low-energy collisions typical of quadrupole basedinstruments the b-series of N-terminal fragments or the y-series ofC-terminal fragments predominate. If doubly charged ions are analysedthen both series are often detected. In general, the y-series ionspredominate over the b-series.

If the isolated peptides used in the method of this invention areC-terminal peptides isolated using DITC glass as discussed above, thepeptides will have a free amine after isolation at their N-terminifacilitating labelling with the labels of this invention. As mentionedabove, these labels may all have the same mass so equivalent peptides ineach sample that is analysed will be shifted in mass by the same amount.CID of these peptides will produce fragments from the labels. Theintensities of the label fragments will allow the relative quantities ofequivalent peptides in each sample to be determined. Covalently linkingthe mass labels of this invention to the N-termini of the isolatedpeptides will shift the masses of the b-series of fragment ions by themass of the label, as long as the charge remains on the label. Since themass of the label used for each sample under analysis is the same, therewill be only one ion series produced for all of the samples as long ascollision induced scission of the labelled peptides takes place in thepeptide backbone. This means that it is possible to identify thelabelled peptides by their fragment ions and for any given peptide therewill be only one fragment series for that peptide, irrespective of thenumber of samples being analysed simultaneously. Fragmentation withinthe labels themselves will produce peaks characteristic of each sample.These peaks will occur in a relatively low mass range (see FIG. 24).With a triple quadrupole instrument, it is preferable to use selectedreaction monitoring to achieve the most sensitive detection of thesepeaks. The relative intensities of these peaks will be indicative of therelative amounts of the source protein, from which the peptide wasderived, in the original samples. In natural peptides, the b-series offragment ions tends to be of lower intensity than the y-series. With anappropriately basic mass label or a “pre-ionised” mass label, comprisingfor example a quaternary ammonium centre, the intensity of the b-seriesof ion fragments may be enhanced. Unfortunately, if C-terminal peptidesare used there is no guarantee that the C-terminal amino acid will bebasic, so the y-series fragment ions may be weak. Determination ofstructural information using the y-series would require that theC-terminus of these peptides carry a basic group or a “pre-ionised”group.

The analysis of proteins by tandem mass spectrometry, particularlymixtures of proteins, is complicated by the “noisiness” of the spectraobtained. Proteins isolated from biological samples are usuallycontaminated with buffering reagents, denaturants and detergents, all ofwhich introduce peaks into the mass spectrum. As a result, there areoften more contamination peaks in the spectrum than peptide peaks, andidentifying peaks that correspond to peptides can be a major problem,especially with small samples of proteins that are difficult to isolate.As a result various methods are used to determine which peaks correspondto peptides before detailed CID analysis is performed. Triple quadrupolebased instruments permit “precursor ion scanning” (see Wilm M. et al.,Anal. Chem. 68(3) 527-33, “Parent ion scans of unseparated peptidemixtures” (1996)). The triple quadrupole is operated in “single reactionmonitoring” mode, in which the first quadrupole scans over the full massrange and each gated ion is subjected to CID in the second quadrupole.The third quadrupole is set to detect only one specific fragment ion,which is usually a characteristic fragment ion from a peptide such asammonium ions. An alternative method used with quadrupole/time-of-flightmass spectrometers scans for doubly charged ions by identifying ionswhich when subjected to CID produce daughter ions with highermass-to-charge ratios than the parent ion. A further method ofidentifying doubly charged ions is to look for sets of peaks in thespectrum which are only 0.5 daltons apart with appropriate intensityratios which would indicate that the ions are the same differing only bythe proportion of ¹³C present in the molecule.

By labelling peptides with the mass labels of this invention, a novelform of precursor ion scanning may be envisaged in which peptide peaksare identified by the presence of fragments corresponding to the masslabels of this invention after subjecting the labelled peptides to CID.In particular, the peptides isolated from each sample by the methods ofthis invention may be labelled with more than one mass label. Anequimolar mixture of a “precursor ion scanning” label which is used inall samples and a sample specific label may be used to label thepeptides in each sample. In this way changes in the level of peptides indifferent samples will not have an adverse effect on the identificationof peptide peaks in a precursor ion scan.

Having identified and selected a peptide ion, it is preferably subjectedto CID. The CID spectra are often quite complex and determining whichpeaks in the CID spectrum correspond to meaningful peptide fragmentseries is a further problem in determining the sequence of a peptide bymass spectrometry. Shevchenko et al., Rapid Commun. Mass Spec. 111015-1024 (1997) describe a further method, which involves treatingproteins for analysis with trypsin in 1:1 ¹⁶O/¹⁸O water. The hydrolysisreaction results in two populations of peptides, the first whoseterminal carboxyl contains ¹⁶O and the second whose terminal carboxylcontains ¹⁸O. Thus for each peptide in the sample the should be a doublepeak of equal intensity for each peptide where the double peak is 2daltons apart. This is complicated slightly by intrinsic peptide isotopepeaks but allows for automated scanning of the CID spectrum fordoublets. The differences in mass between doublets can be determined toidentify the amino acid by the two fragments differ. This method may beapplicable with the methods of this invention if N-terminal peptides areisolated.

1. A set of two or more mass labels, each mass label in the setcomprising a mass marker moiety attached via a cleavable linker to amass normalisation moiety, the mass marker moiety being fragmentationresistant, wherein each mass normalisation moiety ensures that each masslabel in the set has the same aggregate mass as determined by massspectrometry, and wherein each mass marker moiety has a mass differentfrom that of all other mass marker moieties as determined by massspectrometry, and wherein all the mass labels in the set aredistinguishable from each other by mass spectrometry.
 2. A set of masslabels according to claim 1, in which each mass marker moiety in the sethas the same structural core, and each mass normalisation moiety in theset has the same structural core that may be the same or different fromthe structural core of the mass marker moieties, and wherein each masslabel in the set comprises one or more mass adjuster moieties, the massadjuster moieties being attached to or situated within the structuralcore of the mass marker moiety and/or the structural core of the massnormalisation moiety, such that every mass marker moiety in the setcomprises a different number of mass adjuster moieties and every masslabel in the set has the same number of mass adjuster moieties.
 3. A setof mass labels according to claim 2, each mass label in the set havingthe following structure:M(A)_(y)-L-X(A)_(z) wherein M is a mass normalisation moiety, X is amass marker moiety, A is a mass adjuster moiety, L is a cleavablelinker, y and z are integers of 0 or greater, and y+z is an integer of 1or greater.
 4. A set of mass labels according to claim 2 or claim 3,wherein the mass adjuster moiety is selected from: (a) an isotopicsubstituent situated within the structural core of the mass markermoiety and/or within the structural core of the mass normalisationmoiety, and (b) substituent atoms or groups attached to the structuralcore of the mass marker moiety and/or attached to the structural core ofthe mass normalisation moiety.
 5. A set of mass labels according toclaim 4, wherein the mass adjuster moiety is selected from a halogenatom substituent, a methyl group and ²H or ¹³C isotopic substituents. 6.A set of mass labels according to claim 5, wherein the mass adjustermoiety is a fluorine atom substituent.
 7. A set of mass labels accordingto claim 1, wherein the cleavable linker attaching the mass markermoiety to the mass normalisation moiety is a linker cleavable bycollision.
 8. A set of mass labels according to claim 7, wherein thecleavable linker comprises an amide bond.
 9. A set of mass labelsaccording to claim 1, wherein the the mass normalisation moietycomprises a fragmentation resistant group.
 10. A set of mass labelsaccording to claim 9, wherein the mass normalisation moiety comprises aphenyl group.
 11. A set of mass labels according to claim 1, wherein themass marker moiety comprises a pre-ionised group.
 12. A set of masslabels according to claim 11, wherein the mass marker moiety comprisesan N-methyl pyridyl group, or a group selected from the followinggroups: —NH₂, —NR₂, —NR₃ ⁺, —SR₃ ⁺, —SO₃ ⁻, —PO₄ ⁻, —PO₃ ⁻, —CO₂ ⁻—,

wherein R is hydrogen or is a substituted or unsubstituted aliphatic,aromatic, cyclic or heterocyclic group.
 13. A set of mass labelsaccording to claim 3, wherein each of the labels in the set has thefollowing structure:

wherein R is hydrogen or is a substituted or unsubstituted aliphatic,aromatic, cyclic or heterocyclic group; L is a cleavable linker; A is amass adjuster moiety; each p is the same and is an integer of 0 orgreater; each y′ may be the same or different and is an integer of 0-4,the sum of all y′ for any one label being equal to y; each z′ may be thesame or different and is an integer of 0-4, the sum of all z′ for anyone label being equal to z; and y+z is an integer of 1 or greater.
 14. Aset of mass labels according to claim 13, wherein R is H, L is an amidebond, p=0, and A is an F atom.
 15. An array comprising two or more setsof mass labels as defined in claim 1, wherein the aggregate mass of eachof the mass labels of any one set in the array is different from theaggregate mass of each of the mass labels of every other set in thearray.
 16. An array of mass labels according to claim 15, wherein eachmass label in at least one set comprises a mass series modifying groupof the same mass as determined by mass spectrometry, the mass seriesmodifying group in each of the mass labels of any one set having adifferent mass from the mass series modifying groups in each of the masslabels of every other set in the array.
 17. An array of mass labelsaccording to claim 16, wherein the mass series modifying groups areattached to the mass labels such that, upon cleaving the cleavablelinker of the mass labels, the mass series modifying groups becomedetached from the mass marker moieties.
 18. An array of mass labelsaccording to claim 16 or claim 17, wherein the mass series modifyinggroups of each set in the array have the same structural core, and eachmass label of any one set in the array has the same number of massseries modifying groups as the other mass labels of that set, and adifferent number of mass series modifying groups from the mass labels ofevery other set in the array.
 19. An array of mass labels according toclaim 18, each mass label in the array having either of the followingstructures:(S)_(x)-M(A)_(y)-L-X(A)_(z)M(A)_(y)-(S)_(x)-L-X(A)_(z) wherein S is a mass series modifying group;M is a mass normalisation moiety; X is a mass marker moiety; A is a massadjuster moiety; L is a cleavable linker; x is an integer of 0 orgreater; y and z are integers of 0 or greater; and y+z is an integer of1 or greater.
 20. An array of mass labels according to claim 16, whereinthe mass series modifying groups comprise an aryl ether group.
 21. Anarray of mass labels according to claim 19 or claim 20, each mass labelin the array having either of the following structures:

wherein A is a mass adjuster moiety; L is a cleavable linker; y and zare integers of 0 or greater; R is hydrogen or is a substituted orunsubstituted aliphatic, aromatic, cyclic or heterocyclic group; each pis the same and is an integer of 0 or greater; x is an integer of 0 orgreater, x being the same for each mass label in any one set of thearray, and the x of any one set being different from the x of everyother set in the array; each y′ may be the same or different and is aninteger of 0-4, the sum of all y′ for any one label being equal to y,and each z′ may be the same or different and is an integer of 0-4, thesum of all z′ for any one label being equal to z; and y+z is an integerof 1 or greater.
 22. An array of mass labels according to claim 16 orclaim 19, wherein the mass series modifying groups of every set in thearray have the same structural core, the mass series modifying group ofthe mass labels of at least one set comprising one or more mass adjustermoieties, the mass adjuster moieties being attached to or situatedwithin the structural core of the mass series modifying group.
 23. Anarray of mass labels according to claim 22, in which every mass label ofevery set in the array has the same number of mass series modifyinggroups, wherein the mass series modifying group in each mass label ofany one set has the same number of mass adjuster moieties as the massseries modifying groups in every other label of that set, and whereinthe mass series modifying groups in the mass labels of any one set havea different number of mass adjuster moieties from the mass seriesmodifying groups in the labels of every other set in the array.
 24. Anarray of mass labels according to claim 23, wherein each of the sets inthe array comprises mass labels having either of the followingstructures:S(A*)_(r)-M(A)_(y)-L-X(A)_(z)M(A)_(y)-S(A*)_(r)-L-X(A)_(z) wherein S is a mass series modifyinggroup; M is a mass normalisation moiety; X is a mass marker moiety; A isa mass adjuster moiety of the mass marker moieties and massnormalisation moieties; A* may be the same or different from A and is amass adjuster moiety of the mass series modifying groups; L is acleavable linker; r is an integer of 0 or greater and is at least 1 forone or more sets of mass labels in the array; y and z are integers of 0or greater; and y+z is an integer of 1 or greater.
 25. An array of masslabels according to claim 24, wherein each of the sets in the arraycomprises mass labels having either of the following structures:

wherein R is hydrogen or is a substituted or unsubstituted aliphatic,aromatic, cyclic or heterocyclic group; each p is the same and is aninteger of 0 or greater; x is an integer of 0 or greater x being thesame for all mass labels in the array; each y′ may be the same ordifferent and is an integer of 0-4, the sum of all y′ for any one labelbeing equal to y; each z′ may be the same or different and is an integerof 0-4, the sum of all z′ for any one label being equal to z; y+z is aninteger of 1 or greater; each r′ may be the same or different, the sumof all r′ for any one label being equal to r; and r is an integer of 0or greater and is at least 1 for one or more sets of mass labels in thearray.